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IMMUNOBIOLOGY
Killing of human immunodeficiency virus–infected primary T-cell blasts by
autologous natural killer cells is dependent on the ability of the virus to alter
the expression of major histocompatibility complex class I molecules
Matthew I. Bonaparte and Edward Barker
In the current study, we evaluated whether
the capacity of HIV to modulate major
histocompatibility complex (MHC) class I
molecules has an impact on the ability of
autologous natural killer (NK) cells to kill
the HIV-infected cells. Analysis of HIVinfected T-cell blasts revealed that the
decrease in MHC class I molecules on the
infected cell surface was selective. HLA-A
and -B were decreased on cells infected
with HIV strains that could decrease MHC
class I molecules, whereas HLA-C and -E
remained on the surface. Blocking the
interaction between HLA-C and -E and
their corresponding inhibitory receptors
increased NK cell killing of T-cell blasts
infected with HIV strains that reduced
MHC class I molecules. Moreover, we
demonstrate that NK cells lacking HLA-C
and -E inhibitory receptors kill T-cell blasts
infected with HIV strains that decrease
MHC class I molecules. In contrast, NK
cells are incapable of destroying T-cell
blasts infected with HIV strains that were
unable to reduce MHC class I molecules.
These findings suggest that NK cells lacking inhibitory receptors to HLA-C and -E
kill HIV-infected CD4ⴙ T cells, and they
indicate that the capacity of NK cells to
destroy HIV-infected cells depends on the
ability of the virus to modulate MHC class
I molecules. (Blood. 2004;104:2087-2094)
© 2004 by The American Society of Hematology
Introduction
Natural killer (NK) cell–mediated cytotoxic responses seem ideal
for controlling virus-infected cells during the very early stages of
primary HIV infection. NK cells do not require prior recognition of
HIV to eliminate the infected cells, whereas adaptive anti-HIV
responses require prior sensitization and several days to develop.1
Moreover, the adaptive immune response does not reach its optimal
effect until several weeks after infection.2,3 Thus, early effective
restraint of the infected cells by NK cells is needed to keep the
virus under control until the adaptive immune response has a
chance to develop.
Although NK cells are part of the innate arm of the immune
response, they are prevented from killing cells that express major
histocompatibility complex (MHC) class I molecules, which
engage inhibitory molecules on the NK cell surface.4-6 Three
different classes of NK cell inhibitory receptors regulating the
cytotoxic function of NK cells interact with different MHC class I
molecules (for a review, see Borrego et al7). Despite differences in
specificity of the various NK cell inhibitory receptors, they all
contain immunoreceptor tyrosine–based inhibitory motifs (ITIMs)
in their cytoplasmic tails.8-10 The function of ITIMs is to recruit
inhibitory phosphatases, such as Src homology domain–containing
tyrosine phosphatases (SHP)–1 and SHP-2,11-13 which prevent
activation and inhibit NK cell cytotoxicity.14,15 The signal provided
by the inhibitory receptor is dominant over activation signals when
activation and inhibitory molecules are engaged simultaneously.16
Therefore, the presence of MHC class I molecules on the cell
surface serves to inhibit NK cell cytotoxicity.
HIV makes use of several mechanisms to evade the potent
CD8⫹ T-cell response that develops soon after infection.17 One
mechanism by which HIV-infected cells escape recognition and,
thereby, destruction by CD8⫹ cytotoxic T-lymphocytes (CTLs) is
the ability of the virus to decrease MHC class I molecules on
the infected cell surface.18-20 Given that HIV-infected cells avoid
CD8⫹ CTL destruction by down-modulating MHC class I molecules on the cell surface and that NK cells are able to kill cells
with diminished MHC class I molecules, it follows that HIVinfected cells are susceptible to NK cell–mediated killing.
However, we recently demonstrated that NK cells have limited
ability to kill HIV-infected cells despite reduced numbers of
MHC class I molecules.21
A proposed mechanism accounting for the decreased ability of
NK cells to kill HIV-infected cells is the selective reduction of
HLA-A and -B molecules by HIV while it leaves HLA-C and -E on
the infected cell surface.22-25 However, several studies suggest that
HIV may be unable to regulate all NK cells by leaving HLA-C and
-E on the infected cell surface because polyclonal NK cells do not
always coexpress multiple MHC class I–specific inhibitory receptors.26-29 Even in patients with viremic HIV infection, it has been
reported that only 14% of NK cells express inhibitory receptors that
recognize HLA-E.30 Hence, HIV may, at best, be able to prevent
NK cells expressing HLA-C and -E inhibitory receptors from
destroying the infected cells, but not NK cells lacking these
receptors. We determined in our study whether NK cells lacking
inhibitory receptors directed to HLA-C and HLA-E are able to kill
From the Department of Microbiology and Immunology, State University of New
York, Upstate Medical University, Syracuse, NY.
Reprints: Edward Barker, Department of Microbiology and Immunology, State
University of New York, Upstate Medical University, 750 East Adams St,
Syracuse, NY 13210; e-mail: [email protected].
Submitted February 24, 2004; accepted April 20, 2004. Prepublished online as
Blood First Edition Paper, April 29, 2004; DOI 10.1182/blood-2004-02-0696.
Supported by grant AI 52809 from the National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD.
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
2087
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2088
BONAPARTE and BARKER
CD4⫹ T cells infected with HIV in which HLA-A and -B molecules
are decreased.
Materials and methods
Human subjects
After informed consent was obtained, venous blood (50 mL) was acquired from
healthy HIV-uninfected donors into heparinized (250 units; Sigma Chemical, St
Louis, MO) 60-mL syringes (BD Biosciences, Franklin Lakes, NJ). Peripheral
blood mononuclear cells (PBMCs) were isolated from the heparinized blood by
Ficoll-Hypaque gradient (Mediatech, Herndon, VA) centrifugation, as described.31 The institutional review board for the protection of human subjects of
State University of New York Upstate Medical University (Syracuse, NY)
approved the studies described in this article.
HIV infection of CD4ⴙ T cells
Primary CD4⫹ T cells were isolated from PBMCs using anti-CD4 antibodies
coupled to magnetic beads according to the manufacturer’s instructions (Dynal,
Lake Success, NY), resuspended at 3 ⫻ 106 cells/mL in complete T-cell medium,
consisting of RPMI 1640 medium (Mediatech) with 10% heat-inactivated fetal
bovine serum (FBS; Mediatech), 2 mM glutamine (Mediatech), 100 ␮g/mL
streptomycin (Mediatech), 100 U/mL penicillin (Mediatech), and 200 U/mL
recombinant human interleukin-2 (rhIL-2) (obtained from the AIDS Research
and Reference Reagent Program, Division of AIDS, National Institute of Allergy
and Infectious Disease [NIAID], National Institutes of Health [NIH]; rhIL-2 from
Dr Maurice Gatley, Hoffmann-LaRoche, Nutley, NJ)32 and stimulated with 3
␮g/mL phytohemagglutinin (PHA; Sigma) for 3 days. PHA-stimulated CD4⫹ T
cells were shown to be more than 98% CD4⫹ and less than 1% CD14⫹ by flow
cytometry. Stimulated CD4⫹ T cells were spin-infected33 with 1000 ⫻ TCID50
(median tissue culture infective dose) of HIV-1SF162, HIV-1SF128A, HIV-192US712,
HIV-1SF33, or HIV-1SF2 for 5 days, and cells were split 1:2 every 2 days with
fresh, complete T-cell medium. Infected cells were used for the determination of
MHC class I molecule expression by flow cytometry or were processed further
for use as target cells in a chromium 51 (51Cr) release assay.
Flow cytometry
HIV-infected cells or uninfected CD4⫹ T cells (106) were resuspended in
100 ␮L flow cytometry buffer (phosphate-buffered saline [PBS; Mediatech]
with 0.1% NaN3 [Sigma]) and were stained directly with either 2 ␮L
phycoerythrin (PE)–conjugated mouse anti–human pan MHC class I
antibody (clone W6/32; [DAKO Cytomation, Carpinteria, CA]), 5 ␮L
fluorescein isothiocyanate (FITC)– or PE-conjugated mouse anti–human
CD4 antibody (PharMingen, La Jolla, CA), 4 ␮L FITC-conjugated mouse
anti–human HLA-A2 antibody (PharMingen), or 10 ␮L FITC-conjugated
mouse anti–human HLA-B7 antibody (Serotec, Raleigh, NC). To stain cells
for HLA-E, 1 ␮L primary mouse anti–human HLA-E antibody was added
to 106 cells (clone 3D1234; a gift from Dr D.E. Geraghty, Fred Hutchison
Research Institute, Seattle, WA) followed by staining with 1 ␮L biotinconjugated goat anti–mouse immunoglobulin G (IgG) antibody (Caltag,
Burlingame, CA) and 1 ␮L PE-conjugated streptavidin (Caltag). For
staining with anti HLA-C antibody (clone L31; a gift from Dr P. Giacomini,
Regina Elena Cancer Institute, Rome, Italy), 106 cells were treated with 200
␮L citrate-phosphate buffer (pH 3.0) for 2 minutes and were washed twice
with flow cytometry buffer, as described.35 This step is necessary because
the L31 clone of anti–HLA-C antibody recognizes ␤2 microglobulin–less
HLA-C class I MHC heavy chains.36 Acid-treated cells were then surface
stained with 1 ␮L primary mouse anti–human HLA-C antibody, followed by
staining with 2.5 ␮L PE-conjugated goat anti–mouse IgG antibody (Caltag).
Negative controls for cells stained with the specific anti-HLA class I antibody
consisted of either FITC-conjugated, isotype-specific IgG (PharMingen), PEconjugated goat anti–mouse IgG antibody (Caltag) in the absence of primary
antibody or biotin-conjugated goat anti–mouse IgG antibody, and PE-conjugated
streptavidin (Caltag) in the absence of primary antibody.
All surface staining was performed on ice for 20 minutes, and cells were
washed twice with 1 mL flow cytometry buffer between each staining step.
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
Cells were then fixed and permeabilized using the Cytofix/Cytoperm kit
(PharMingen), resuspended in 100 ␮L Perm-Wash buffer (PharMingen),
and stained with 5 ␮L human anti-HIV p24 capsid antibody (obtained from
the AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH: monoclonal antibody to HIV-1 p24 [clone 71-31] from Dr
Susan Zolla-Pazner, New York University, New York, NY),37 followed by 2
␮L FITC-conjugated goat anti–human IgG antibody (Beckman Coulter,
Miami, FL) or 10 ␮L PE-conjugated goat anti–human IgG antibody
(Caltag). All intracellular staining was performed for 20 minutes on ice, and
cells were washed twice with 1 mL Perm-Wash buffer between each
staining step. As negative controls for anti-HIV p24 antibody staining, cells
were treated with secondary antibody only. Infected cells (104 HIV-1 p24
antigen-positive) or uninfected CD4⫹ cells (104) were acquired using a flow
cytometer (LSR-II; BDIS, San Jose, CA) and the level of CD4 or MHC
class I molecules on the cell surface was determined using CellQuest
Software (BDIS).
To determine the expression of NK cell inhibitory receptors specific for
HLA-C and HLA-E on NK cells, 2 ⫻ 106 PBMCs were resuspended in 200
␮L flow cytometry buffer, and 7.5 ␮L PC5-conjugated anti-CD56 antibody
(Beckman Coulter), 15 ␮L PE-conjugated anti-CD158a antibody (Beckman
Coulter), 15 ␮L PE-conjugated anti-CD158b antibody (Beckman Coulter),
and 15 ␮L FITC-conjugated CD94 antibody (PharMingen) were added to
the cell suspension for 20 minutes on ice. As a negative control, 10 ␮L each
isotype control antibody (PharMingen) was added to a suspension of
PBMCs for 20 minutes on ice. Cells were washed twice in flow cytometry
buffer and fixed in 500 ␮L PBS with 1% paraformaldehyde (pH 7.4).
CD56⫹ cells (104) were acquired with a flow cytometer (LSR-II; BDIS),
and the percentage of CD56⫹ cells stained with various inhibitory receptors
was determined using CellQuest Software (BDIS).
Isolation of NK cells and their subpopulations lacking
HLA-C– and HLA-E–specific inhibitory receptors
NK cells were isolated from PBMCs obtained from the same HIVuninfected subjects who donated blood for the generation of HIV-infected
T-cell blasts. NK cells were acquired using anti-CD56 antibody coupled to
magnetic beads, per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). This separation technique resulted in a
population of cells that was more than 90% CD56⫹ (data not shown). NK
cells were resuspended in RPMI 1640 medium with 10% heat-inactivated
FBS, 2 mM glutamine, 100 ␮g/mL streptomycin, and 100 U/mL penicillin
for the 51Cr release assay, added in triplicate in 100-␮L aliquots to a 96-well
round-bottom plate (Falcon, Franklin Park, NJ) at the proper effector to
target cell (E/T) ratios, and placed at 37°C in a 5% CO2 humidified
incubator overnight.
Two different approaches were used to isolate the NK cell subpopulations lacking inhibitory receptors to HLA-C and -E. In one study (Figures 5,
6A-B), NK cells were first isolated from PBMCs using anti-CD56 antibody
coupled to magnetic beads and a rare earth magnet (StemCell Technologies). CD56⫹ cells were then depleted of CD94⫹, CD158a⫹, and CD158b⫹
cells (Figures 5 and 6A), or CD159a⫹, CD158a⫹, and CD158b⫹ (Figure
6B) by adding 0.75 ␮g each purified mouse antibody (Beckman Coulter)
per 106 isolated CD56⫹ cells for 30 minutes at 4°C. Antibody-labeled cells
were incubated with sheep anti–mouse IgG magnetic beads according to the
manufacturer’s instructions (Dynal) and were depleted of inhibitory
receptor–positive cells using a magnet provided by the manufacturer
(Dynal). In another study (Figure 6C-D), PBMCs were depleted of
CD158a⫹, CD158b⫹, and CD94⫹ cells by negative selection using 15 ␮L
each of PE-conjugated CD158a, CD158b, and CD94 antibodies (Beckman
Coulter) per 106 inhibitory receptor–positive PBMCs. PE-positive cells
were negatively selected using the EasySep PE selection cocktail according
to the manufacturer’s instructions (StemCell Technologies). NK cells were
then isolated from the CD158a-, CD158b-, and CD94-depleted PBMCs
(negative fraction) using magnetic beads coupled to anti-CD56 antibody
and a rare earth magnet (StemCell Technologies) according to the manufacturer’s instructions. The CD158a/b- and CD94- or CD159a-depleted NK
cells were shown to be routinely more than 90% CD56⫹ and less than 5%
CD158a/b⫹ and CD94⫹ or CD158a/b⫹ and CD159a⫹ by flow cytometry
(data not shown). No difference in percentage of CD94⫹ and CD159a⫹ NK
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
cells was observed, regardless of the method used to deplete inhibitory
receptor–positive cells from the NK cells (data not shown). The inhibitory
receptor–depleted NK cells were added in 100-␮L aliquots to a 96-well
round-bottom plate in RPMI 1640 medium with 10% heat-inactivated FBS,
2 mM glutamine, 100 ␮g/mL streptomycin, and 100 U/mL penicillin and
were placed in a 37°C incubator with 5% CO2 overnight before use in the
cytotoxicity assay.
Isolation of HIV-infected primary T-cell blasts for use
in the NK cell cytotoxic assay
HIV-infected primary T-cell blasts used as target cells in the 51Cr release
assay were isolated as described previously.21 Briefly, the HIV-infected
cells were separated from the uninfected cells using the method described,38
which is based on the observation that CD4 molecules are down-modulated
on HIV-infected cells. Uninfected CD4⫹ T cells were removed with
anti-CD4 antibody-conjugated to magnetic beads (Dynal), thus enriching
for cells containing HIV (Figure 1). This method allows for the normalization of cells harboring HIV regardless of the percentage of HIV-infected
cells acquired after 5-day infection of CD4⫹ cells with HIV. Cells
remaining after separation were less than 1% CD4⫹ (Figure 1A). The CD4⫺
cells were shown to express HIV p24 antigen (Figure 1B). If the HIV strain
reduced MHC class I molecules, the CD4⫺ and HIV p24 antigen-positive
cells had decreased numbers of MHC class I molecules (Figure 1C-D,
respectively [mean fluorescence intensity [MFI], 261.34 for uninfected
CD4⫹ to 56.63 for HIV-infected or CD4⫺ cells]). HIV-infected (CD4⫺)
cells were then washed 3 times in calcium- and magnesium-free Hanks
balanced salt solution (HBSS) and used as target cells in a 51Cr release assay.
NK cell cytotoxicity assay
NK-sensitive K562 human erythroleukemia cells (American Tissue Culture
Collection, Manassas, VA), uninfected CD4⫹ T-cell blasts, and HIV-
Figure 1. Expression of CD4, HIV p24 antigen, and MHC class I molecules on
HIV-infected primary CD4ⴚ T-cell blasts. HIV-1SF162–infected primary T-cell blasts
were depleted of CD4⫹ cells before their use as target cells in the cytotoxicity assay.
CD4-depleted HIV-infected cells (bold lines) were stained with fluorochromeconjugated anti-CD4 antibody (A,C) or human anti-HIV p24 antibody, together with
fluorochrome-conjugated goat anti–human IgG antibody (B,D). Cells were then
stained with fluorochrome-conjugated anti-MHC class I antibody (C-D). As controls,
uninfected CD4⫹ T cells (thin solid lines) were also stained with the same antibody.
MHC class I molecules expressed on 104 uninfected CD4⫹ control cells (C-D), 104
HIV p24 antigen-positive cells (D), and 104 CD4⫺ cells (C) were determined. Dotted
lines indicate isotype control antibody (Ab).
REGULATION OF NK CELLS BY HIV IS STRAIN DEPENDENT
2089
infected T-cell blasts were used as target cells in a 4-hour 51Cr release assay,
as described.21 K562 cells were labeled with 100 ␮Ci (3.7 MBq) 51Cr
(Amersham Biosciences, Piscataway, NJ) per 106 cells for 2 hours, and
uninfected CD4⫹ T-cell blasts and HIV-infected T cells were labeled with
125 ␮Ci (4.63 MBq) 51Cr per 106 cells for 2 hours.
When NK cell inhibitory receptors to HLA-C and -E were blocked
(Figure 4), 10 ␮g/mL purified anti-CD158a, CD158b, and CD159a
antibody (Beckman Coulter) was added to the NK cells before target cells
were added. The percentage of specific lysis was calculated using the
formula: [(sample cpm ⫺ mean spontaneous release cpm)/(mean maximal
release cpm ⫺ mean spontaneous release cpm)] ⫻ 100. The cpm of
spontaneous release was typically 10% to 20% of the cpm of maximal
release. The 2-tailed Student t test was used to determine statistical
differences between the treatment and the control groups.
Results
Ability of HIV to modulate MHC class I molecules on primary
T-cell blasts is strain dependent
In this study, we determined whether different HIV strains were
able to decrease MHC class I molecules on primary CD4⫹ T cells.
We evaluated the expression of MHC class I molecules using an
antibody (clone W6/32) that recognized a conserved epitope found
on all MHC class I molecules. During peak virus production, after
5-day infection with 1000 ⫻ TCID50 of HIV-1SF162, HIV-1SF33,
HIV-1SF128A, HIV-1SF2, and the primary HIV isolate HIV-192US712,
we determined the extent to which MHC class I molecules were
expressed. When we evaluated the expression of MHC class I
molecules on HIV-infected (HIV p24 antigen-positive) T-cell
blasts, we found that HIV-1SF162 was able to decrease MHC class I
molecules on the cell surface (mean fluorescence intensity [MFI],
68.55) relative to the MHC class I molecules on uninfected CD4⫹ T
cells (MFI, 100.72) (Figure 2A). In contrast to HIV-1SF162–infected
cells, primary T-cell blasts infected with HIV-1SF33 (MFI, 110.77)
had levels of MHC class I molecules similar to those found in the
uninfected controls (MFI, 100.72) (Figure 2A). It should be noted
that although there was no decrease in MHC class I molecules on
HIV-1SF33–infected cells, there was a decrease in the percentage of
infected cells expressing CD4 molecules and an increase in the
percentage of HIV p24 antigen-positive cells compared with HIV-1SF162–
infected cells (data not shown). Thus, the failure of HIV-1SF33 to
decrease MHC class I molecules was not the result of inadequate
infection but was inherent in the virus strain used for infection.
We found that the primary T-cell blasts infected with HIV-1SF128A
(Figure 2B) and the primary HIV isolate HIV-192US712 (Figure 2C) had
decreased expression of MHC class I molecules on the cell surface
relative to the MHC class I molecules found on uninfected cells. In
contrast, MHC class I molecules on HIV-1SF2–infected cells were not
decreased (Figure 2B). Thus, some strains of HIV down-modulate
MHC class I molecules on primary T-cell blasts, whereas other strains
are unable to alter MHC class I expression.
HLA-A and -B, but not HLA-C and -E, molecules are decreased
on primary T-cell blasts infected with HIV strains
that decrease MHC class I molecules
Because some HIV strains decrease MHC class I molecules, we
determined whether the reduction in MHC class I molecules was
selective or whether all MHC class I molecules were modulated. For this
reason, we evaluated the expression of HLA-A, -B, -C, and -E on
primary CD4⫹ T cells infected with HIV-1SF162 or HIV-1SF33. We
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2090
BONAPARTE and BARKER
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
Figure 2. Expression of MHC class I molecules on primary T-cell blasts after infection with various strains of HIV. PHA-stimulated primary CD4⫹ T cells from
HIV-uninfected donors were infected with HIV-1SF162 (A, C), HIV-1SF33 (A), HIV-1SF128A (B), HIV-1SF2 (B), or HIV-192US712 (C) for 5 days. Cells were stained with PE-conjugated
anti-MHC class I antibody followed by human anti-HIV p24 antibody, together with FITC-conjugated goat anti–human IgG antibody. As controls, uninfected CD4⫹ T cells were
also stained with the same antibody. The extent to which 104 HIV p24 antigen-positive infected cells and uninfected cells expressed MHC class I molecules on the cell surface
was determined.
evaluated the HIV p24 antigen-positive cells for expression of the
various HLA-class I molecules during peak virus production (5 days
after infection). As seen in Figure 3, HIV decreased the expression of
HLA-A2 on HIV-1SF162–infected (HIV p24⫹) T-cell blasts (MFI,
109.72) relative to uninfected CD4⫹ T cells (MFI, 218.79) (Figure 3A).
Reduction of HLA-B7 was also observed on HIV-1SF162–infected (HIV
p24⫹) cells (MFI, 5.70) compared with uninfected cells (MFI, 45.87)
(Figure 3B). In contrast to the decreased levels of HLA-A and -B
molecules on HIV-infected cells compared with uninfected cells,
HLA-C and HLA-E on HIV-infected (HIV p24⫹) cells (MFI, 68.00 and
116.77, for HLA-C and -E, respectively) were not reduced relative to
uninfected controls (MFI, 37.08 and 108.86, for HLA-C and -E,
respectively [Figure 3C-D]). In contrast to HIV-1SF162–infected
cells, we did not observe modulation of any type of HLA class I
molecules on HIV-1SF33–infected cells (p24⫹) compared with
uninfected cells (Figure 3E-H). Thus, when HIV decreased MHC
Figure 3. Expression of various types of MHC class I molecules on T-cell blasts infected with different HIV strains. HIV-1SF162–infected (A-D) and HIV-1SF33–infected
(E-H) T-cell blasts were stained with human anti-HIVp24 capsid antibody (Ab) and FITC- or PE-conjugated goat anti–human IgG antibody. Levels of HLA-A2 (A,E), -B7 (B,F),
-C (C,G), and -E (D,H) on the surfaces of infected cells (HIV-1 p24 antigen-positive) and uninfected cells were determined after flow cytometric analysis of cells stained with the
corresponding mouse anti–human HLA class I antibody. Histograms were gated on 104 infected HIV p24 antigen-positive cells (heavy solid lines) and were compared with 104
uninfected CD4⫹ cells (solid lines). Results are representative of 3 separate experiments. Negative controls (dashed lines) consisted of either FITC-conjugated isotype-specific
IgG (A-B, E-F), PE-conjugated goat anti–mouse IgG antibody in the absence of primary antibody (C,G) or biotin-conjugated goat anti–mouse IgG antibody and PE-conjugated
streptavidin (D,H) in the absence of primary antibody.
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
class I molecules, the reduction was selective for HLA-A and -B
but not for HLA-C and -E.
Blocking CD158a, CD158b, and CD159a on NK cells from
interacting with HLA-C and -E molecules on HIV-infected cells
increases the ability of NK cells to kill the infected cells
Given that HLA-C and -E prevent NK cells from destroying
healthy cells,39-41 we determined whether polyclonal NK cells
could kill HIV-infected cells directly when the NK cells were
treated with blocking antibody to CD158a, CD158b, and CD159a.
Although the heterodimer of CD94 and CD159a recognizes
HLA-E, the ITIM motifs, important for inhibitory signaling, are
contained on the CD159a molecule.40 Hence, in our study, only the
interaction between CD159a and HLA-E was blocked. As expected,21 little if any killing of HIV-infected T cells by NK cells
occurred, though the same NK cells could kill the NK-sensitive
K562 cells (Figure 4). An increase in NK cell–mediated killing of
HIV-infected cells was observed (P ⬍ .05) when they were treated
with antibody to the inhibitory receptors before target cells were
added to the effector cells (Figure 4). Although NK cells killed
HIV-infected cells when HLA-C and -E inhibitory receptors were
blocked, the killing was limited to 15.7% at an E/T ratio of 30:1 and
was not statistically different from the response at an E/T ratio of
5:1 (P ⱖ .3) (Figure 4B-C).
Determining whether NK cells lacking inhibitory receptors for
HLA-C and -E kill HIV-infected autologous primary T-cell blasts
Given that blocking HLA-C and -E and their inhibitory receptors
increased NK cell killing (Figure 4), we sought to determine
REGULATION OF NK CELLS BY HIV IS STRAIN DEPENDENT
2091
whether NK cells lacking inhibitory receptors to HLA-C and -E are
able to destroy autologous CD4⫹ T cells infected with HIV-1SF162
and HIV-1SF33. We initially evaluated 8 subjects’ peripheral blood
NK cells for the expression of inhibitory receptors recognizing
HLA-C and -E (Table 1). Either CD158a or CD158b inhibitory
receptor recognizes HLA-C molecules. CD94 is paired with
CD159a as an inhibitory receptor recognizing HLA-E.40,41 Because
CD94 is the marker that appeared to be expressed on the NK cells
the least, and because between 92% and 98% of CD159a⫹ NK cells
were CD94⫹ (data not shown), we evaluated CD94 as a marker for
NK cell inhibitory receptors for HLA-E. In Table 1, we illustrate
that of the 8 subjects evaluated, on average only 10.31% of the NK
cells expressed inhibitory receptors that recognized HLA-C and -E
molecules. Within the NK cell population, on average 19.44%
expressed inhibitory receptors for HLA-C but not HLA-E, and
39.31% expressed inhibitory receptors for HLA-E but not HLA-C.
However, 30.93% of the NK cells did not express CD94, CD158a,
or CD158b receptors. Therefore, not all NK cells found in the
peripheral blood express inhibitory receptors known to bind to
HLA-C and -E.
HLA-A and -B were decreased on HIV-1SF162–infected cells
(Figure 3A-B), and HLA-C and -E remained on the cell surface
(Figure 3C-D); therefore, we wanted to determine whether isolating NK cells lacking CD158a, CD158b, and CD94/CD159a
increased the ability of the remaining NK cells to mediate the
killing of HIV-infected primary T-cell blasts. By removing NK
cells that expressed inhibitory receptors for HLA-C and -E, we
would enrich for NK cells that would not likely be inhibited by
HLA-C and -E and, thus, would destroy those cells infected with
HIV that would modulate HLA-A and -B but not HLA-C and -E.
Figure 4. Blocking the interaction between CD158a, CD158b, and CD159a on NK cells and HLA-C and -E molecules on infected cells increases the ability of NK cells
to destroy HIV-infected autologous primary T cells. HIV-1SF162–infected T-cell blasts, uninfected CD4⫹ T cells, and K562 cells were used as target cells in a 4-hour
cytotoxicity assay. Effector cells (NK cells) were isolated from the same donor from whom the CD4⫹ T cells were isolated and were incubated with or without 10 ␮g/mL purified
anti-CD158a, CD158b, and CD159a antibody before addition to target cells. Effector cell–target cell ratios of 1:1, 5:1, 10:1, and 30:1 were used. All samples were performed in
triplicate. The Student t test was used to determine the statistical differences between percentage of specific lysis of HIV-infected cells by NK cells in the absence or presence of
anti-CD158, -CD158b, and -CD159a antibody. *P ⬍ .05; **P ⬍ .01. ND indicates not done.
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2092
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
BONAPARTE and BARKER
Table 1. Distribution of NK cells expressing inhibitory receptors for HLA-C and HLA-E
Subject
CD158a/bⴙCD94ⴚ*
CD158a/bⴙCD94ⴙ*
CD158a/bⴚCD94ⴚ*
CD158a/bⴚCD94ⴙ*
1
14.28
13.22
28.77
43.72
2
26.62
7.91
33.08
32.38
3
25.52
16.42
23.94
34.12
4
12.26
14.85
33.16
39.73
5
18.73
7.52
36.07
37.68
6
19.34
4.92
29.65
46.08
7
23.74
11.54
27.81
36.91
8
14.99
6.16
34.95
43.89
Mean ⫾ SD
19.44 ⫾ 5.06
10.31 ⫾ 3.99
30.93 ⫾ 3.83
39.31 ⫾ 4.61
PBMCs were isolated from 8 healthy HIV-uninfected subjects and were stained with a PE cyanin 5.1-conjugated antibody directed to the NK cell marker CD56,
PE-conjugated anti-CD158a and -CD158b antibodies, and FITC-conjugated anti-CD94 antibodies.
Mean ⫾ SD values represent those of the subpopulations of NK cells from all subjects tested.
*Percentages of 104 CD56⫹ cells expressing CD94, CD158a, and CD158b molecules were determined.
For this purpose, we evaluated the ability of NK cells lacking
CD158a, CD158b, and CD159a/CD94 to destroy autologous
HIV-1SF162–infected cells in a 4-hour 51Cr release assay. NK cells
from 2 different donors increased killing of HIV-1SF162–infected
cells relative to unfractionated controls (Figure 5).
Given that HIV-1SF162 decreases HLA-A and -B but leaves
HLA-C and -E on the surface (Figure 3) and that HIV-1SF33 was
unable to decrease any of the MHC class I molecules (Figure 3),
we compared the ability of NK cells lacking HLA-C and -E
receptors to kill HIV-1SF162–infected cells and HIV-1SF33–
infected cells. NK cells from 2 different donors lacking CD158a,
CD158b, and CD159a/CD94 killed HIV-1SF162 infected cells by
16.5% (Figure 6A) and 10.8% (Figure 6B) at a 5:1 E/T ratio and
by 20.7% (Figure 6A) and 16.5% (Figure 6B) at a 10:1 E/T ratio.
In contrast, NK cells had little, if any, ability to kill HIV-1SF33–
infected cells despite the ability of the same NK cells to kill the
NK cell–sensitive cell line K562 (Figure 6C-D). These studies
indicate that the strain of HIV determines the susceptibility of
HIV-infected cells to killing by NK cells lacking inhibitory
receptors directed at HLA-C and -E.
recently demonstrated that X4 strain HIV-1SF13 does not decrease
MHC class I molecules (M.I.B. and E.B., unpublished observations, November 2003). The R5X4 strain HIV-1SF2 was unable to
decrease MHC class I molecules (Figure 2). Whether this pattern of
modulating MHC class I molecules is consistent for other R5, X4,
and R5X4 HIV strains remains to be determined. We observed that
a primary R5 isolate, HIV-192US712 (Figure 2C), also decreased
MHC class I molecules, indicating that reducing MHC class I
molecules is an ability not limited to laboratory-adapted strains.
The dominant HIV strains in HIV-infected asymptomatic persons
are R5 strains.42 On the contrary, HIV isolated from persons whose
disease progresses to AIDS typically has a broad range of
coreceptor (ie, R5 and X4) usage, but the strains isolated from
AIDS patients are typically X4 strains.42 Therefore, it would be of
interest to determine the ability of primary NK cells to control
autologous HIV-infected T cells from infected persons at various
stages of disease.
When the different types of MHC class I molecules were
evaluated on infected cells, HIV appeared to selectively decrease
Discussion
The HIV strains in our study, HIV-1SF162 and HIV-1SF128A, which
decreased MHC class I molecules on infected cells (Figure 2), used
the CCR5 coreceptor (R5) for viral entry. In contrast, HIV strains
(eg, HIV-1SF33) that do not decrease MHC class I molecules (Figure
2) use the CXCR4 coreceptor (X4) for viral entry. Furthermore, we
Figure 5. NK cells lacking CD158a, CD158b, and CD159a have greater ability to
kill HIV-infected cells. HIV-1SF162–infected T-cell blasts were used as target cells in a
4-hour cytotoxic assay. NK cells were depleted of CD158a⫹, CD158b⫹, and
CD159a⫹ cells before their use as effector cells in the cytotoxic assay. NK cells not
depleted of CD158a⫹, CD158b⫹, and CD159a⫹ cells were used as controls. Effector
cell–target cell ratios of 1:1, 5:1, and 10:1 were used. All samples were performed in
triplicate. Student t test was used to determine the statistical differences between
percentage of specific lysis of HIV-infected cells by NK cells depleted of cells bearing
CD158a, CD158b, and CD159a, with percentage of specific lysis of HIV-infected cells
by unfractionated NK cells containing CD158⫹, CD158b⫹, and CD159a⫹ cells.
*P ⬍ .05; **P ⬍ .01
Figure 6. NK cells lacking CD158a, CD158b, and CD94 or CD159a kill autologous T-cell blasts infected with an HIV strain that decreases MHC class I
molecules but not T-cell blasts infected with an HIV strain that does not alter
MHC class I molecules. HIV-1SF162–infected T-cell blasts (A-B), HIV-SF33–infected
T-cell blasts (C-D), and K562 cells (A-D) were used as target cells in a 4-hour
cytotoxic assay. NK cells were depleted of CD158a⫹, CD158b⫹, and CD94⫹ or
CD159a⫹ cells before their use as effector cells in the cytotoxic assay. Effector
cell–target cell ratios of 1:1, 2.5:1, 5:1, and 10:1 were used. All samples were
performed in triplicate.
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
HLA-A and -B but not HLA-C (Figure 3A-C). Moreover, the
nonclassical HLA-E molecules remained on the surface of the
infected cell (Figure 3D). These observations were similar to
those of other investigators, who demonstrated that HIV infection led to decreased HLA-A and -B expression but not HLA-C
or -E expression.25 It should be noted that this previous study
used infectious HIV clones and cell lines in which the CD4 and
specific MHC class I molecules were stably transfected. However, our studies used primary PHA-stimulated CD4⫹ T cells
infected with different strains of intact virus originally isolated
from infected subjects. In addition, infected cells with relatively
high levels of HIV p24 antigen expressed within the cell had
greater reductions in HLA-A and -B molecules on the cell
surface than infected cells with relatively low levels of HIV p24
antigen (data not shown).
Studies by others indicate that NK cells are unable to destroy
HIV-infected cells because HLA-E and -C decrease NK cell
responses by receiving inhibitory signals through CD94/CD159a,
CD158a, or CD158b.25 However, we demonstrated that only some
NK cells expressed inhibitory receptors to HLA-C and -E (Table 1).
Furthermore, when we blocked the interaction between the MHC
class I molecules and their receptors (Figure 4), we observed
increased killing of HIV-infected T-cell blasts by NK cells. Hence,
some NK cells may be, but are not exclusively, under the control of
HLA-C and -E molecules on HIV-infected cells.
The rate of killing by unfractionated NK cells is at best 6.3%.
Therefore, it is difficult, even at an E/T ratio of 30:1 (Figure 4), to
observe killing of HIV-infected cells in the 51Cr release assay if the
number of NK cells lacking these receptors make up approximately
30% of the population (Table 1). Enriching a subpopulation of NK
cells that lacked inhibitory receptors to HLA-C and -E allowed us
to determine whether the remaining NK cells were able to kill
HIV-infected cells (Figures 5-6). We observed killing of HIVinfected cells with NK cells lacking HLA-C and -E inhibitory
receptors (P ⬍ .05) when the virus used to infect CD4⫹ cells
decreased MHC class I molecules.
The strain of HIV infecting CD4⫹ T cells determines the
susceptibility of the infected cells to destruction by NK cells. When
the T-cell blasts were infected with an HIV strain that decreased
HLA-A and -B (Figure 3A-B), NK cells lacking CD94/CD159a,
CD158a, and CD158b were able to destroy the HIV-infected cells
(Figure 5A-B) to a greater degree than unfractionated NK cells. In
contrast, when HLA-A and -B were not decreased (Figure 3E-F),
NK cells lacking CD94/CD159a, CD158a, and CD158b were
unable to kill the infected cells (Figure 6C-D).
When we exposed the HIV-1SF33–infected cells to anti-HIV
gp120 antibody, we observed at best 16.2% lysis of the infected
cells by autologous NK cells at an E/T ratio of 10:1, even though
98% of the target cells were HIV-1 p24 antigen-positive (data not
shown). No killing of HIV-1SF33–infected cells by NK cells was
observed in the absence of anti-gp120 antibody in this same
experiment. Thus, even though the killing of the X4 virus–infected
cells by autologous NK cells in the presence of anti-HIV gp120
antibody (ie, antibody dependent cell–mediated cytotoxicity) is
relatively low, it is comparable to previously observed levels of
killing of HIV-1SF162–infected cells in Figure 6 (20.7% lysis).
Moreover, other investigators43 have also shown approximately
30% specific lysis of HIV-1IIIB (X4)–infected autologous primary T
cells by HIV antigen–specific CTL lines.
The variability in the number of HIV-1–infected cells and
NK cells lacking HLA-C and HLA-E inhibitory receptors
acquired from a subject limited the E/T ratios to 5:1 and
REGULATION OF NK CELLS BY HIV IS STRAIN DEPENDENT
2093
sometimes 10:1. However, we observed increased lysis of the
HIV-infected autologous T cells by CD158 a/b and CD159a/
CD94 NK cells over the unfractionated population of NK cells,
even at relatively low E/T ratios (Figure 5). The relatively low
level of NK cell killing of HIV-infected T cells (compared with
the killing of K562 cells) was not attributed to a low number of
HIV-infected target cells, because the target cells were normalized for CD4-negative–infected cells that were HIV p24 antigenpositive (Figure 1). The best rate we observed of specific lysis of
HIV-infected cells by autologous NK cells was 20.7%, at an E/T
ratio of 10:1 (Figure 6). This level is nowhere near that observed
for NK cell killing of K562 cells. Even though it is difficult to
determine whether the level of killing observed in our study was
physiologically relevant, NK cells that play a role in controlling
herpes virus infection in humans44 can kill herpesvirus-infected
autologous cells at a percentage of specific lysis of 30% to 40%
when using NK cell clones as effector cells.45,46
Primary T-cell blasts infected with HIV strains that decrease
MHC class I molecules are susceptible to killing by NK cells
lacking receptors to HLA-C and -E. However, the killing of these
infected cells may not be optimal. Based on our current study, NK
cells appear to be important in the control of HIV strains that
modulate MHC class I molecules (Figure 6) and may be responsible for curtailing the capacity of the virus to replicate when CTL
responses are unable to do so.22,47 However, these responses appear
to be limited to NK cells lacking inhibitory receptors specific to
HLA-C and -E (Figures 4-6). Even when enriched for cells lacking
CD158a/b and CD159a/CD94, the extent to which these NK cells
destroyed HIV-infected cells, even under ideal conditions, appears
to have been limited (Figures 5-6). NK cell killing of HIV-infected
cells in the presence of blocking antibody was not greater with an
E/T ratio of 30:1 than it was with an E/T ratio of 5:1 (Figure 4).
Increased killing did not result from the ability of anti-CD158a,
CD158b, or CD159a antibody to bind to the infected cell surface
and to mediate antibody-dependent, cell-mediated cytotoxicity
because we were unable to observe the expression of these
molecules on the surfaces of infected T cells or uninfected CD4⫹
cells.48 Thus, there may be a limit to which NK cells lacking
inhibitory receptors to HLA-C and -E can kill HIV-infected cells.
It may be that NK cells lacking inhibitory receptors to HLA-C
and -E do not completely kill HIV-infected T lymphocytes because
HIV can induce the expression of another nonclassical MHC class I
molecule, HLA-G. HLA-G molecule expression on extravillous
cytotrophoblasts is important during pregnancy because this molecule modulates maternal NK responses.49 Recent studies from our
laboratory demonstrate that HIV can induce the expression of
HLA-G on the HIV-infected T-cell surface though uninfected
CD4⫹ T lymphocytes do not express HLA-G (M.I.B. and E.B.,
unpublished observations, June 2002). Approximately 65% of
HIV-1SF162–infected cells expressed HLA-G, whereas approximately 95% of HIV-1SF33–infected cells expressed this nonclassical
MHC class I molecule. Unlike the limited expression of HLA-A,
-B, -C, and -E–specific inhibitory receptors on NK cells, almost all
NK cells express the HLA-G–specific receptor CD158d,50,51 and
most NK cells express the HLA-G inhibitory receptor, CD85j.52
Thus, HLA-G may limit NK cells lacking CD94/CD159a, CD158a
and/or CD158b, or both from killing autologous HIV-infected
primary T-cell blasts (Figures 5-6) by triggering CD158d and
CD85j. Further studies to evaluate the role of HLA-G in regulating
NK cell responses to HIV-infected cells are under way.
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2094
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
BONAPARTE and BARKER
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2004 104: 2087-2094
doi:10.1182/blood-2004-02-0696 originally published online
April 29, 2004
Killing of human immunodeficiency virus-infected primary T-cell blasts
by autologous natural killer cells is dependent on the ability of the virus
to alter the expression of major histocompatibility complex class I
molecules
Matthew I. Bonaparte and Edward Barker
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