The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 12, November 26, 2007 3027-3036 www.jem.org/cgi/doi/
Despite signifi cant advances over the past two
decades with respect to our understanding of the
immune response to HIV-1 infection, the precise
immune correlates of protection against HIV-1
disease progression are still largely unknown.
Epidemiological studies have identifi ed several
host genetic factors that are strongly associated
with better HIV-1 disease outcome. A 32 – base-
pair deletion in CC chemokine receptor (CCR)
5 is associated with an increased resistance to
infection ( 1 – 3 ). Furthermore, a signifi cant pro-
tective eff ect from HIV-1 disease progression
has been described for several human leukocyte
antigen (HLA) class I alleles, whereas an asso-
ciation with more rapid disease progression
has been described for other HLA class I alleles
( 4 – 6 ). These studies suggest a critical role for
HIV-1 – specifi c CD8 + T cells in the control of
viral replication and have helped guide func-
tional studies in the area of T cell immunology
in HIV-1 infection. More recently, a signifi cant
association has been described between slower
HIV-1 disease progression and the expression
of an activating NK cell receptor, the killer
immunoglobulin-like receptor (KIR) 3DS1, in
conjunction with its putative ligand, HLA-B
Bw4 alleles with an isoleucine at position 80
(referred to as HLA-B Bw4-80I) ( 7 ). This pro-
tective eff ect was independent of the expression
of the respective HLA-B Bw4-80I alleles HLA-
B57 or HLA-B27 ( 7 ). These epidemiological
data were the fi rst to implicate specifi c NK cell
receptor genes in modulating HIV-1 patho-
genesis, but the functional basis of the epidemi-
ological association between these KIR/HLA
compound genotypes and control of HIV-1
replication and disease progression have not yet
been identifi ed.
NK cells are the primary eff ector cells of the
innate immune response, as they are able to lyse
virally infected cells and secrete large amounts of
proinfl ammatory cytokines/chemokines without
Abbreviations used: CCR, CC
chemokine receptor; E/T,
eff ector/target; HLA, human
leukocyte antigen; KIR, killer
MFI, mean fl uorescence inten-
sity; SSO, thianthrene 5-oxide.
The online version of this article contains supplemental material.
Diff erential natural killer cell – mediated
inhibition of HIV-1 replication based
on distinct KIR/HLA subtypes
Galit Alter, 1 Maureen P. Martin, 2 Nickolas Teigen, 1 William H. Carr, 1
Todd J. Suscovich, 1 Arne Schneidewind, 1 Hendrik Streeck, 1
Michael Waring, 1 Angela Meier, 1 Christian Brander, 1 Jeff rey D. Lifson, 3
Todd M. Allen, 1 Mary Carrington, 2 and Marcus Altfeld 1
1 Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital and Division of AIDS,
Harvard Medical School, Boston, MA 02129
2 Laboratory of Genomic Diversity and 3 Retroviral Pathogenesis Section, Basic Research Program, SAIC-Frederick, Inc.,
Frederick, MD 21702
Decline of peak viremia during acute HIV-1 infection occurs before the development of
vigorous adaptive immunity, and the level of decline correlates inversely with the rate of
AIDS progression, implicating a potential role for the innate immune response in determining
disease outcome. The combined expression of an activating natural killer (NK) cell receptor,
the killer immunoglobulin-like receptor (KIR) 3DS1, and its presumed ligand, human
leukocyte antigen (HLA) – B Bw4-80I, has been associated in epidemiological studies with a
slow progression to AIDS. We examined the functional ability of NK cells to differentially
control HIV-1 replication in vitro based on their KIR and HLA types. NK cells expressing
KIR3DS1 showed strong, signifi cant dose- and cell contact – dependent inhibition of HIV-1
replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not
express KIR3DS1. Furthermore, KIR3DS1 + NK cells and NKLs were preferentially activated,
and lysed HIV-1 infected target cells in an HLA-B Bw4-80I – dependent manner. These data
provide the fi rst functional evidence that variation at the KIR locus infl uences the effec-
tiveness of NK cell activity in the containment of viral replication.
NK CELL – MEDIATED INHIBITION OF HIV-1 | Alter et al.
evaluations of this in vitro system demonstrated that freshly
isolated NK cells were able to inhibit HIV-1 replication in
autologous HIV-1 – infected CD4 + T cells. NK cells persisted
throughout the 14-d culture and acquired an activated pheno-
type expressing high levels of CD56, KIR, NKp46, CD161,
and NKG2D (Fig. S1, available at http://www.jem.org/cgi/
Results for the inhibition of viral replication at diff erent E/T
ratios showed high concordance when tested in three sepa-
rate experiments on cells derived from the same individuals
(small standard deviations), as demonstrated for a representa-
tive individual in Fig. 1 A . Furthermore, NK cell – mediated
inhibition of HIV-1 replication in autologous CD4 + T cells
was dependent on the number of NK cells added into CD4 +
T cell cultures ( Fig. 1, A and B ), further suggesting that NK
cells are directly responsible for the inhibition of HIV-1 rep-
lication observed in this system. The level of viral replication
peaked by day 7 after infection, and at that time the strongest
NK cell – mediated inhibition of HIV-1 replication was ob-
served, expressed as the log diff erence in p24 Gag produc-
tion between CD4 + T cells alone or in the presence of NK
cells ( Fig. 1, A and B ). Collectively, these data demonstrate
that the ability of NK cells to inhibit HIV-1 replication can
be quantifi ed in vitro and that the inhibition of viral replica-
tion depends on the NK eff ector cell/CD4 + T cell target
NK cells suppress the spread of HIV-1 infection in vitro
Although the production of p24 Gag is a strong surrogate
marker for the level of viral replication in vitro, it does not
allow for the determination of whether the observed suppres-
sion of p24 Gag production is caused by the continuing in-
fection of CD4 + T cell targets but reduced virion release, or
by the reduction of the number of infected CD4 + T cells.
We used intracellular p24 staining on infected T cells at 3 – 4-d
intervals to quantify the number of infected cells over time
and observed a signifi cantly lower proportion of p24 + T cells
in wells containing NK cells and CD4 + T cells at an E/T ratio
of 10:1 compared with wells containing HIV-1 – infected CD4 +
T cells alone or NK cells at lower E/T ratios ( Fig. 1 C and
Fig. S2, available at http://www.jem.org/cgi/content/full/jem
.20070695/DC1). Given that only NK cells (CD3 − CD56 +/ ?
CD16 +/ ? ) and CD4 + T cells (CD3 + CD4 + ) cells were present
in the co-cultures, we stained cells with CD3 to distinguish
these two populations and assessed the level of p24 in cells
expressing CD3. Overall, p24 Gag production quantifi ed in
the supernatant by ELISA was signifi cantly correlated to the
percentage of p24 + CD3 + T cells quantifi ed by fl ow cyto-
metry (r= 0.66; P = 0.002). These results demonstrate that
the observed NK cell – mediated inhibition of HIV-1 replica-
tion was caused by the containment of HIV-1 infection to a
small subset of CD4 + T cells.
NK cells require contact to mediate HIV-1 inhibition
NK cells are involved in the control of viral infection both via
the release of antiviral cytokines and the direct lysis of target
prior antigen sensitization ( 8 ). NK cells play a critical role in
the control of viral replication in several disease models ( 9 – 11 ),
and depletions or defi ciencies of NK cells lead to signifi -
cantly more severe viral disease and death in both mice and
humans ( 12, 13 ). In addition to the protective eff ect of the
KIR3DS1/HLA-B Bw4-80I compound genotype in HIV-1
infection ( 7 ), the protective eff ects of particular KIR/HLA
genotypes have been demonstrated in several other epidemi-
ological studies in human viral infections, including hepatitis
C virus ( 14 ), human papillomavirus ( 15 ), and CMV reactiva-
tion after transplantation ( 16 ), suggesting that the interaction
between KIRs and their HLA class I ligands can have a pro-
found impact on the effi cacy of NK cell – mediated control of
viral pathogenesis ( 17 ).
As many as 15 diff erent KIRs have been identifi ed ( 18 ),
and this family of glycoprotein receptors expressed predomi-
nantly on NK cells can be grouped into categories based on
whether they possess two or three immunoglobulin-like
domains ( 18 ). They are also characterized by long or short cyto-
plasmic tails that transmit inhibitory or activating signals,
respectively ( 18 ). KIR3DS1 has been recently shown to be
expressed on the surface of NK cells ( 19, 20 ) and to associate
with Dap12 ( 19 ), a characteristic of activating NK cell recep-
tors. Although the precise ligand for KIR3DS1 has not been
identifi ed, HLA class I molecules of the HLA-B Bw4-80I
family have been proposed as putative ligands, as KIR3DS1
shares > 97% sequence homology in the extracellular domains
with KIR3DL1, an inhibitory KIR that interacts with several
HLA-B Bw4-80I molecules ( 18, 21 – 24 ).
We hypothesized that the observed epidemiological asso-
ciation of the KIR3DS1/HLA-B Bw4-80I genotype with
slower HIV-1 disease progression is caused by the ability of
KIR3DS1 + NK cells to strongly inhibit HIV-1 replication in
HLA-B Bw4-80I + CD4 + T cells, and we developed an in vitro
viral replication inhibition assay to test this hypothesis. In this
paper, we demonstrate that NK cells are able to suppress
HIV-1 replication in vitro in a dose- and cell contact – dependent
manner. Furthermore, we show that NK cells expressing
KIR3DS1 strongly and signifi cantly inhibited HIV-1 replica-
tion in target cells expressing HLA-B Bw4-80I relative to NK
cells that do not express KIR3DS1. These data provide the
fi rst functional evidence that variation at the KIR locus infl u-
ences the eff ectiveness of NK cell activity in the containment
of HIV-1 replication.
NK cells can suppress HIV-1 replication in vitro
To determine the anti-HIV-1 activity of NK cells in vitro,
we developed a co-culture assay to measure the level of HIV-1
replication in CD4 + T cells in the presence or absence of
autologous NK cells. In this assay, purifi ed activated CD4 + T
cells were infected with HIV-1 and coincubated with puri-
fi ed autologous NK cells at diff erent eff ector/target (E/T) ra-
tios over a period of 14 d in the presence of IL-2, and HIV-1
replication was quantifi ed using a p24 Gag ELISA. The initial
JEM VOL. 204, November 26, 2007
cells and HIV-1 – infected CD4 + T cells were in contact
during co-culture was added on days 3, 7, 10, and 14 to wells
containing only autologous HIV-1 – infected CD4 + T cells.
Supernatant alone mediated some viral suppression over the
course of the 14-d period ( Fig. 1 E ), albeit at signifi cantly
lower levels than in wells where the cells were in direct con-
tact. Collectively, these data demonstrate that direct cell-to-
cell contact between NK cells and autologous HIV-1 – infected
CD4 + T cells is required to trigger the inhibition of HIV-1
replication in this system, and that secreted factors alone are
not suffi cient to mediate signifi cant inhibition.
Subjects expressing KIR3DS1 and HLA-B Bw4-80I suppress
HIV-1 replication most potently compared with individuals
expressing only one or none of these alleles
To assess the impact of the KIR/HLA compound geno-
type on the ability of NK cells to inhibit HIV-1 replication
in vitro, NK cell – mediated inhibition of viral replication
was quantifi ed in a group of 36 HIV-1 – negative study subjects
expressing a diverse array of KIR/HLA genotypes. As demon-
strated in Fig. 2 A , the level of NK cell – mediated inhibition
of HIV-1 replication in autologous CD4 + T cells in vitro
diff ered largely among the 36 subjects tested, ranging from 0
to 2.1 logs at day 7, when inhibition of viral replication was
To test the initial hypothesis that NK cells derived from
individuals with the KIR3DS1/HLA-B Bw4-80I compound
genotype mediate strong antiviral activity, the study sub-
jects were grouped according to their genotype ( KIR3DS1/
Bw4-80I , n = 8; KIR3DS1 alone, n = 8; Bw4 alone, n = 10;
and neither gene, n = 10). As early as day 3, NK cells derived
from subjects possessing both KIR3DS1 and HLA-B Bw4-
80I alleles exhibited a superior level of viral inhibition com-
pared with NK cells derived from subjects who had one of
the two or neither of the alleles ( Fig. 2 B ); these diff erences
reached statistical signifi cance at days 7, 10, and 14 (P < 0.01
for comparisons of each group versus KIR3DS1 + /Bw4-80I + ).
In contrast, the ability of HIV-1 to replicate in CD4 + T cells
in the absence of NK cells did not diff er across the four geno-
typic groups (P > 0.3 for comparisons of p24 Gag levels be-
tween each group versus KIR3DS1 + /Bw4-80I + ; Fig. S2 C),
demonstrating that the inhibition of viral replication was
caused by diff erences in the antiviral activity of the added
NK cells and not by diff erences in the ability of the virus to
replicate in the diff erent CD4 + T cell populations studied.
Data presented in Fig. 2 were derived from experiments
using a CCR5-tropic HIV-1 strain derived from a clinical
sample; however, similar suppressive activity was observed
with CCR5-tropic laboratory-adapted strains and CXCR4-
tropic strains (unpublished data). These data demonstrate that
NK cells derived from subjects with the compound genotype
KIR3DS1/HLA-B Bw4-80I exhibit strong anti – HIV-1 ac-
tivity in vitro and for the fi rst time provide functional data in
support of a plausible mechanistic basis for the described epi-
demiological association between this genotype and slower
HIV-1 disease progression.
cells ( 25 ). To determine whether NK cells require contact
with infected cells to mediate viral inhibition, NK cells and
HIV-1 – infected CD4 + T cells were separated in transwell
experiments at an E/T ratio of 10:1. No signifi cant inhibi-
tion of HIV-1 replication was observed when NK cells and
auto logous infected CD4 + T cells were separated by a semiper-
meable membrane ( Fig. 1 D ), demonstrating that direct cell-
to-cell contact was required for the initial triggering of NK
cell activity and/or for mediating their antiviral activity.
Furthermore, to determine whether secreted factors from
NK cells alone were suffi cient to inhibit viral replication
in vitro, supernatant collected from experiments in which NK
Figure 1. NK cells are able to mediate inhibition of HIV-1 replica-
tion in a contact-dependent manner. (A) The line graph depicts the
mean change in the viral load (log10 p24 Gag in supernatant) observed in
a single subject from three separate experiments in wells containing CD4 +
T cells alone (continuous black line), and NK cells and autologous CD4 + T
cells at a ratio of 10:1 (short dashed black line), 1:1 (long dashed gray
line), and 1:10 (long dashed black line). (B) The fi gure represents a log
reduction in p24 Gag production at different NK cell/CD4 + T cell ratios
(10:1, 1:1, and 1:10) for days 3, 7, 10, and 14 ( n = 5). (C) The whisker box
plots show the percentage of p24 Gag + CD3 + T cells at days 3, 7, 10, and
14 in wells containing NK cells at different NK cell/CD4 + T cell ratios (10:1, 1:1,
and 1:10; n = 6). (D) The fi gure represents the log inhibition of HIV-1
replication observed on days 3, 7, 10, and 14 in wells where NK cells and
autologous HIV-1 – infected CD4 + T cells were co-cultured together (gray)
or separated in a transwell experiment (black) at an NK cell/CD4 + T cell
ratio of 10:1 ( n = 5). (E) The whisker box plots show the log inhibition of
HIV-1 replication observed on days 3, 7, 10, and 14 in wells where NK cells
and autologous HIV-1 – infected CD4 + T cells were co-cultured together
(gray) at an NK cell/CD4 + T cell ratio of 10:1, or where the supernatant
was transferred from co-cultured cells to autologous HIV-1 – infected
CD4 + T cells (black; n = 5). Data represent the mean of at least three ex-
periments and standard deviations.
NK CELL – MEDIATED INHIBITION OF HIV-1 | Alter et al.
(a) NK cells that express neither KIR3DS1 nor KIR3DL1
(z27 neg DX9 neg ; Fig. 3 ), (b) NK cells expressing KIR3DS1
alone (z27 lo DX9 neg ; Fig. 3 ), and (c) NK cells expressing
KIR3DL1 alone or in conjunction with KIR3DS1 (z27 hi DX9 pos ,
as both of these populations are included in the medium-high
MFI population [ 19 ]; Fig. 3 A ).
The respective NK cell populations were individually
sorted from PBMCs derived from fi ve HLA-B Bw4-80I + in-
dividuals expressing both KIR3DS1 and KIR3DL1 using an
extreme gating strategy to guarantee the sorting of highly puri-
fi ed subpopulations of NK cells ( Fig. 3 A ), and they were used
separately as eff ector cells in a viral replication inhibition assay
with autologous HIV-1 – infected CD4 + T cells at an E/T ratio
of 10:1. Notably, inhibition of HIV-1 replication mediated by
the KIR3DS1 pos KIR3DL1 neg NK cell populations was signifi -
cantly greater than that mediated by the KIR3DL1 pos subset of
NK cells (1.48 vs. 0.3 logs; P = 0.01; Fig. 3 B ). The KIR3DS1 pos
subset also mediated greater viral inhibition than the KIR3D-
S1 neg KIR3DL1 neg NK cells (1.48 vs. 0.79 logs; P = 0.04).
KIR3DS1 + NK cells are mainly responsible for the
suppression of HIV-1 replication
KIR molecules are expressed in a stochastic, variegated man-
ner on NK cells within a given individual, giving rise to NK
cell clones with distinct KIR expression profi les in that indi-
vidual ( 26 ). This results in the presence of both KIR3DS1 pos
and KIR3DS1 neg NK cell populations in an individual who
has the allele encoding for KIR3DS1. To determine whether
the KIR3DS1 pos NK cell populations from subjects express-
ing KIR3DS1 and HLA-B Bw4-80I were responsible for the
observed in vitro suppression of HIV-1 replication, we used
two monoclonal antibodies — one directed against KIR3DL1
(DX9) and one directed against both KIR3DL1 and KIR3DS1
(z27) ( 19 ) — to sort the individual NK cell populations ex vivo.
Although the DX9 antibody only labels KIR3DL1 pos NK
cells, z27 labels both KIR3DS1 pos NK cells with a low mean
fl uorescence intensity (MFI) and KIR3DL1 pos NK cells with a
high MFI ( 19, 27, 28 ). The combination of these antibodies
with distinct specifi cities allowed for the dissection of three sep-
arate NK cell populations, as previously described ( 19, 20, 29 ):
Figure 3. NK cells expressing KIR3DS1 inhibit HIV-1 replication
more effectively and respond stronger to Bw4-80I + HIV-1 – infected
target cells. (A) The fl ow plots demonstrate the segregation of KIR3DL1 +
NK cells (1), KIR3DS1 + /KIR3DL1 − NK cells (2), and NK cells expressing neither
of these receptors (3), using the combination of the z27 and DX9 antibodies
for a subject that does not encode KIR3DS1 (left) and one that does encode
KIR3DS1 (right). Numbers represent the proportion of events present within
that quadrant. (B). The dot plot represents the log inhibition of HIV-1 repli-
cation (given as log inhibition of p24 Gag production) in co-cultures of
autologous HIV-1 – infected Bw4-80I + CD4 + T cells with bulk NK cells, sorted
populations of KIR3DS1 + /3DL1 − NK cells, KIR3DL1 + NK cells, and NK cells
expressing neither of these markers at an NK cell/CD4 + T cell ratio of 10:1.
All experiments were performed in triplicate in fi ve different KIR3DS1 + /
KIR3DL1 + subjects. Horizontal bars represent the mean log inhibition.
Figure 2. NK cells derived from subjects with KIR3DS1 and HLA-B
Bw4-80I suppress viral replication more effectively in vitro. (A) The
dot plot demonstrates the heterogeneity of viral suppression (given as log
inhibition of p24 Gag production) observed in the total cohort of 36 study
subjects at an NK cell/CD4 + T cell ratio of 10:1 ( n = 36). Horizontal bars
represent the mean log inhibition. (B) The whisker box plots represent the
log inhibition observed in subjects with KIR3DS1 and HLA-B Bw4-80I
(black; n = 8), HLA-Bw4 alone (light gray; n = 10), KIR3DS1 alone (white;
n = 8), and individuals that express neither of these two alleles (dark gray;
n = 10), where NK cells and CD4 + T cells were co-cultured at an E/T ratio
of 10:1. Data represent the mean and SDs. **, P < 0.001; *, P < 0.05.
JEM VOL. 204, November 26, 2007
HIV-1 – infected Bw4 + CD4 + T cells, we monitored KIR3DS1 pos /
3DL1 neg and KIR3DL1 pos NK cell degranulation in bulk NK
cells derived from heterozygous KIR3DL1 + /3DS1 + individ-
uals ( 30 ) after 7 d of co-culture with autologous Bw4 + or
Bw6 + HIV-1 – infected CD4 + T cells. CD107a expression
was increased fi vefold in KIR3DS1 pos /3DL1 neg NK cells
after exposure to HIV-1 – infected Bw4 + CD4 + T cells but
increased less than twofold after coincubation with HIV-1 –
infected Bw6 + CD4 + T cells (P = 0.03; Fig. 4, A and B ).
In contrast, very little increase in CD107a expression was
observed on KIR3DL1 pos NK cells after coincubation with
autologous HIV-1 – infected CD4 + T cells irrespective of the
HLA-B Bw genotype of the individual ( Fig. 4 B , bottom).
KIR3DL1 + NK cells responded more potently to the MHC-
devoid target cells K562 or 221 than to KIR3DS1 + NK cells,
suggesting that the diff erence observed after stimulation with
HIV-1 – infected autologous target cells was specifi c to the
Furthermore, the NK cell population that did not contain
KIR3DS1 + NK cells (the KIR3DL1 − /3DS1 − population)
inhibited HIV-1 replication to a lesser extent than bulk NK
cells (0.8 vs. 1.33 logs; P = 0.06; Fig. 3 B ). The residual anti-
viral activity of the KIR3DS1 neg KIR3DL1 neg NK cells sug-
gests that other NK cell receptors in addition to KIR3DS1
are also able to restrict HIV-1 replication to some extent,
specifi cally in the absence of KIR3DL1. Overall, these data
demonstrate that KIR3DS1 pos NK cells are largely responsi-
ble for the NK cell – mediated suppression of in vitro HIV-1
replication observed in individuals expressing the KIR3DS1/
HLA-B Bw4-80I genotype.
KIR3DS1 + NK cells preferentially lyse HIV-1 – infected
HLA-B Bw4-80I – expressing target cells
To further evaluate whether NK cells expressing KIR3DS1
in the absence of KIR3DL1 were preferentially activated by
Figure 4. KIR3DS1 + NK cells are activated and lyse HIV-1 – infected cells in the presence of HLA-B Bw4-80I alleles. (A) Using the combination
staining of DX9 and z27, gates were set along the z27 axis to gauge the level of degranulation on z27 hi (DX9 + z27 + or KIR3DL1 + ), z27 dim (DX9 − z27 + or
KIR3DS1 + ), or z27 neg (DX9 − z27 − or double negative) NK cells after stimulation with (a) medium alone, (b) K562, (c) 221 cells, (d) uninfected autologous
CD4 + T cells, (e) autologous HIV + CD4 + T cells, and (f) PMA/ionomycin. NK cells and target cells were plated at a ratio of 10:1 for all experiments. (B) The
line graphs show the percentage of CD107a + NK cells derived from KIR3DS1 + /HLA-B Bw4 + (black lines) and KIR3DS1 + /HLA-B Bw6 + (gray lines) individuals
after the culture of NK cell populations in the presence of autologous HIV-1 – uninfected or – infected CD4 + T cells (at a ratio of 10:1). Gating strategies
were used to calculate the percentage of CD107a + NK cells within the KIR3DS1 + NK cell population (z27 + DX9 − ; top) or within the KIR3DL1 + NK cell popu-
lation (z27 + DX9 + ; bottom). Only KIR3DS1 + /3DL1 − NK cells co-cultured with Bw4 + HIV-1 – infected CD4 + T cells signifi cantly (P = 0.03) up-regulated CD107a
expression. (C) The whisker box plot represents the percentage of untransfected 221 cells that were lysed in a 6-h chromium assay using bulk NK cells
derived from KIR3DS1 + subjects (light gray; n = 6) and KIR3DS1 − individuals (dark gray; n = 6). NK cells were co-cultured with Cr 51 -labeled targets at a
ratio of 20:1. (D) The bar graph displays the ratio of the percent lysis of HIV-1 – infected versus – noninfected, HLA-transfected 221 cells (with 52% of cells
staining for p24 by intracellular fl ow at the maximal time of infection) by a KIR3DS1-transfected NKL (NKL/221 target cell, 20:1; n = 3). Data represent
the mean of at least three experiments and standard deviations.
NK CELL – MEDIATED INHIBITION OF HIV-1 | Alter et al.
on HIV-1 disease progression ( 7 ), implicating NK cells directly
in the control of HIV-1 pathogenesis.
The expression of specifi c KIRs in conjunction with their
HLA ligands has been shown to have an impact on disease
outcome in several epidemiological studies in diff erent human
viral infections, including protection against HIV ( 7, 31 ),
hepatitis C virus ( 14 ), CMV reactivation ( 16 ), and, more re-
cently, protection against seroconversion in a group of female
sex workers that were repeatedly exposed to HIV but re-
mained seronegative ( 32 ). However, it is still unclear from
these epidemiological studies what mechanism accounts for
NK cell – mediated control of viral replication. In this paper,
we demonstrate that NK cells are able to suppress HIV-1 viral
replication in vitro. Furthermore, we show that KIR3DS1 +
NK cells exert their antiviral activity preferentially in the
presence of HLA-B Bw4-80I on HIV-1 – infected target cells.
Collectively, these data provide the fi rst functional evidence
that NK cells can contribute directly to the control of HIV-1
replication in a receptor – ligand – specifi c manner.
KIRs are stochastically expressed on NK cells. Thus, in a
given KIR3DS1 + /KIR3DL1 + individual, a subset of NK cell
clones expresses KIR3DS1 alone, a small subset may express
KIR3DS1 in combination with KIR3DL1 ( 20 ), and others do
not express KIR3DS1 at all. To address whether KIR3DS1 +
NK cells preferentially mediated the inhibitory eff ect on
HIV-1 replication observed for subjects that encode this re-
ceptor, we used a gating strategy based on the combination of
two KIR3DS1/KIR3DL1-specifi c antibodies (DX9 and z27)
( 19, 20, 27 – 29 ) to sort populations of NK cells that expressed
KIR3DS1 and not KIR3DL1 (DX9 − and z27 lo ), KIR3DL1
(DX9 + and z27 hi ), or neither of these two KIRs (DX9 − and
z27 − ). Thus, using these individual NK cell popu lations derived
from the same individual, we demonstrate that KIR3DS1 + /
KIR3DL1 − NK cells were signifi cantly more potent in me-
diating inhibition of HIV-1 replication relative to the other
NK cell subpopulations. Furthermore, the NK cell popula-
tion that did not include KIR3DS1 + NK cells (KIR3DL1 − /
3DS1 − ) inhibited HIV-1 replication to a lesser extent than
bulk NK cells. The observation that KIR3DS1 − /KIR3DL1 −
NK cells were also able to mediate some viral inhibition
(albeit at lower levels than KIR3DS1 + NK cells) was in
line with previous studies demonstrating that additional NK
cell receptors, including natural cytotoxicity receptors, may
also be involved in the recognition of HIV-1 – infected target
cells ( 33, 34 ).
In contrast, little inhibition of HIV-1 replication was medi-
ated by NK cell populations expressing the inhibitory receptor
KIR3DL1 (DX9 + and z27 hi ) despite the fact that these DX9/
z27 double-positive NK cells may also include a small fraction
of NK cells that express both KIR3DL1 and KIR3DS1 ( 20 ).
Recent data demonstrate that particular KIR3DL1 haplotypes
are in fact associated with slower HIV-1 disease progression
( 31 ), suggesting that specifi c KIR3DL1 subtypes may also be
critically involved in the recognition of HIV-1 – infected target
cells, perhaps by monitoring for changes in the downmodula-
tion of MHC class I alleles by HIV Nef. Further studies are
KIR3DS1 + NK cell subpopulation ( Fig. 4 A ). To eliminate
the possibility that bulk NK cell populations derived from
subjects that express KIR3DS1 were just intrinsically more
potent at recognizing target cells, we further measured the
lytic activity of bulk NK cell populations against HLA- devoid
chromium-labeled 221 cells. There was no diff erence in the
level of killing of 221 cells among subjects that possessed or
did not possess the KIR3DS1 gene ( Fig. 4 C ). Collectively,
these data demonstrate that KIR3DS1 + NK cells can recog-
nize HIV-1 – infected HLA-Bw4 + CD4 + T cells.
To further evaluate the ability of KIR3DS1 + cells to rec-
ognize HIV-1 – infected target cells in the context of HLA-B
Bw4-80I, we infected 221 cells that were either untrans-
fected or transfected with individual HLA class I alleles
with a pseudotyped R5 virus (JRCSF). HIV-1 – infected and
– uninfected 221 cells were chromium labeled and placed
in co-culture with the NK tumor cell line NKL express-
ing either KIR3DS1, KIR3DL1, or neither of the receptors
for 6 h ( 19, 21 ). The level of virus-associated target cell
lysis by the eff ector NK cells was calculated as a ratio of the
percent lysis of HIV-1 – infected versus – uninfected HLA-
transfected 221 cells. Target cell lysis, irrespective of the
expressed HLA class I allotype, did not signifi cantly diff er
between infected and uninfected 221 cells when exposed
to KIR3DL1 + or KIR3DL1 neg /KIR3DS1 neg NKLs (mean
ratios of lysis of infected/uninfected cells were 1.01, 0.82,
and 0.98 for KIR3DL1 + NKLs, and 0.93, 0.88, and 1.01
for KIR3DL1 neg /KIR3DS1 neg NKLs with respect to the lysis
of HLA-B Bw4-80I – , Bw4-80T – , and Bw6- transfected 221
cells, respectively). Furthermore, there was no increase in
the level of killing by KIR3DS1 + NKLs of HIV-1 – infected
221 cells transfected with either Bw4-80T or Bw6 compared
with uninfected 221 cells ( Fig. 4 D ). In contrast, KIR3DS1 +
NKLs lysed HIV-1 – infected 221 cells expressing HLA-B
Bw4-80I signifi cantly more effi ciently than uninfected Bw4-
80I + 221 cells ( Fig. 4 D and Fig. S3, available at http://
these data using both primary NK cells and KIR-transfected
NKLs demonstrate that KIR3DS1 + NK cells can recog-
nize and lyse HIV-1 – infected target cells in the context of
NK cells play a crucial role in the control of several human
viral infections, but their functional role in HIV-1 pathogen-
esis is not known. In this paper, we demonstrate that NK
cells expressing KIR3DS1, which has recently been shown to
be expressed on the surface of NK cells ( 20, 29 ) and to confer
activating signals to NK cells ( 19 ), are strongly activated by
autologous HIV-1 – infected Bw4-80I + CD4 + T cells and can
signifi cantly inhibit HIV-1 replication in these cells in vitro
compared with NK cells expressing the inhibitory receptor
KIR3DL1, or neither of these receptors. These data provide
the fi rst functional correlation and a plausible mechanistic basis
for the epidemiological studies that described a protective ef-
fect of the KIR3DS1/HLA-B Bw4-80I compound genotype
JEM VOL. 204, November 26, 2007
required to investigate the infl uence of these additional NK
cell receptors on the quality of KIR3DS1-mediated protection
from HIV-1 disease.
To date, immunological studies in HIV-1 infection have
largely focused on virus-specifi c T cell and antibody re-
sponses. Our functional data suggest that, in addition to these
adaptive immune responses, NK cells can contribute to the
antiviral immunity against HIV-1, as suggested by recent epi-
demiological studies ( 7, 31 ). NK cells have been shown to
play a critical role in the initial containment of viral replica-
tion in several viral disease models ( 25, 45 ). In acute HIV-1
infection, NK cells are signifi cantly expanded before the
development of vigorous adaptive immunity ( 46 ), indicating
a potential role for the innate immune response in the initial
control of viral replication. Studies aimed at investigating the
role of CD8 + T cells in the control of SIV/HIV infection
demonstrated a lack of control over viral replication in rhesus
macaques depleted of CD8 + cells, and the results from these
studies were interpreted as demonstrating that CD8 + T cells
are critical for control over SIV replication ( 47, 48 ). However,
the antibody used for these studies, CM-T807 ( 47, 48 ), de-
pleted all cells expressing CD8 on their surface, as acknowledged
by the authors. In monkeys, > 80% of NK cells express CD8,
resulting in their depletion in this experimental system. Thus,
it is possible that not only the depletion of CD8 + T cells
alone but also of NK cells might have been responsible for
or contributed to the loss of control of HIV-1 replication ob-
served in these studies.
In conclusion, a better understanding of the factors that
contribute to the control of HIV-1 replication and the speed
of HIV-1 disease progression is needed to guide the design of
eff ective immunotherapeutic interventions or vaccines. In this
paper, we show compelling evidence that NK cells can limit
HIV-1 replication in vitro and provide an initial functional
correlate for the protective eff ect of the KIR3DS1/HLA-B
Bw4-80I compound genotype from HIV-1 disease progression
described in epidemiological studies ( 7 ). These data suggest a
more important role of eff ector cells of the innate immune
system in the control of HIV-1 replication than traditionally
thought, which will help to guide the development of anti-
viral strategies directly targeting innate immunity.
MATERIALS AND METHODS
Subjects. 36 HIV-1 – negative subjects were recruited for this study. The
study was approved by the local Institutional Review Boards of the partici-
pating institutions, and all individuals gave informed consent for participa-
tion in this study.
HLA and KIR typing. Genomic DNA was extracted from whole PBMCs
and was typed for both KIR and HLA class I alleles. In brief, HLA typing
was performed after amplifi cation of genomic DNA using locus-specifi c
primers that fl anked exons 2 and 3. The PCR products were blotted on
nylon membranes and hybridized with sequence-specifi c oligonucleotide
(thianthrene 5-oxide [SSO]) probes. We assigned alleles by the reaction pat-
terns of the SSO probes. Ambiguous SSO probe typing results were resolved
by sequencing analysis, as previously described ( 5 ). For KIR typing, genomic
DNA was typed for the presence or absence of KIR3DL1 and KIR3DS1
by PCR with sequence-specifi c priming. PCR amplifi cation was performed
required to determine the underlying mechanism of the pro-
tective eff ect of high expressing KIR3DL1 with HLA-B Bw4
80I alleles in HIV-1 disease progression.
In line with the better in vitro inhibition of viral replica-
tion, KIR3DS1 + NK cells were preferentially activated by
HIV-1 – infected Bw4-80I + target cells, because NK cells ex-
pressing KIR3DS1 (DX9 neg z27 lo ) specifi cally degranulated
in the presence of HIV-1 – infected autologous CD4 + T cells.
Furthermore, only KIR3DS1-transfected NKLs and not
KIR3DL1 or untransfected NKLs preferentially lysed HIV-1 –
infected Bw4-80I + cells, but not uninfected Bw4-80I + cells.
How might KIR3DS1 + NK cells recognize HIV-1 – infected
CD4 + T cells? The activating NK cell receptor KIR3DS1
and the inhibitory receptor KIR3DL1 segregate as alleles of
the same gene. KIR3DL1 binds to HLA-B molecules with
the Bw4 motif and preferentially to the Bw4-80I subset of
alleles ( 21, 22 ). Allele-specifi c, as well as peptide-dependent,
binding between HLA class I molecules from the Bw4-80I
family and KIR3DL1 have been recently described in the
context of several diff erent HLA class I epitopes derived from
HIV-1 ( 22, 35 ). KIR3DS1 and KIR3DL1 share ? 97% amino-
acid similarity in their extracellular domains ( 7 ) and may,
therefore, also share a similar set of ligands, as described for
other families of activating and inhibitory KIRs ( 18, 35 – 37 ).
The observation that KIR3DS1 + NK cell populations, as well as
KIR3DS1-transfected NKLs, responded to HLA-B Bw4-80I +
HIV-infected target cells raises the possibility that specifi c modi-
fi cations of the HLA-B Bw4 80I – peptide complex occur during
HIV-1 infection, leading to KIR3DS1 recognition/activation.
Several reports have demonstrated that amino-acid changes
at positions 7 and 8 within an epitope can have a profound
eff ect on inhibitory KIR recognition and, thus, NK cell acti-
vation ( 38 – 40 ). Along the same line, it is possible that activat-
ing KIR signaling may also be infl uenced by specifi c peptides
within the HLA-binding groove. Thus, it is plausible that viral
or stress peptides produced during HIV-1 infection may be
recognized by KIR3DS1 in the context of HLA-B Bw4-80I
molecules, either directly or in conjunction with other re-
ceptors ( 41 ).
Although individuals with the KIR3DS1/HLA-B Bw4
80I haplotype suppressed HIV-1 replication more eff ectively
in vitro than subjects that expressed only one or neither of
these two alleles, there was considerable heterogeneity in NK
cell – mediated viral inhibition among these individuals. One
potential explanation for this heterogeneity is that the quality
of the KIR3DS1 + NK cell – mediated inhibition of HIV-1
might be signifi cantly modulated by the presence of addi-
tional receptors expressed on these NK cells ( 33, 34 ). Our
data show that KIR3DS1 + /3DL1 neg express and transcribe
additional inhibitory KIR and NKG2A (Fig. S4, available at
expression of these additional self-recognizing NK cell recep-
tors, as well as natural cytotoxicity receptors, is necessary for
the effi cient education/licensing of these NK cells ( 42, 43 ),
as well as their cross talk with DCs ( 44 ), both of which ulti-
mately modulate their antiviral activity. Larger studies are
NK CELL – MEDIATED INHIBITION OF HIV-1 | Alter et al.
Cell lysis assay. The lytic activity of NKLs against 221 target cells was as-
sessed by a standard chromium release assay, as previously described ( 49 ).
Thus, 2 × 10 6 221 cells were labeled with 50 ? Ci Na 2 ( 51 CrO 4 ) (1 Ci = 37
GBq; New England Nuclear) for 1 h at 37 ° C, 5% CO 2 . Target cells were
washed three times and plated with eff ector NKLs at an E/T ratio of 20:1.
Supernatant was harvested after a 6-h incubation at 37 ° C, 5% CO 2 . The
percent lysis was calculated as follows: ([sample count − spontaneous release]/
[maximal release − spontaneous release]) × 100.
Statistical analysis. All experiments represent the mean of at least three
experiments and standard deviations. To test for diff erences in the mean
between several populations, a one-way analysis of variance with a Tukey ’ s
correction was used for all comparisons that were below P < 0.05. P < 0.05
was considered signifi cant.
Online supplemental material. Fig. S1 shows data on NK cell numbers
and activation markers over the 14-d co-culture period. Fig. S2 depicts the
gating strategy used for the identifi cation of p24 + T cells (A), the loss of
p24 + T cells in wells containing autologous NK cells (B), as well as the
overall p24 levels in infected T cells among HLA-KIR compound geno-
types (C). Fig. S3 demonstrates the raw data for the NKL lysis assay pre-
sented in Fig. 4 D . Fig. S4 shows the protein (A) and mRNA (B) expression
of additional inhibitory NK cell receptors in bulk NK cells and sorted sub-
populations. Online supplemental material is available at http://www.jem
We thank Dan McVicar, Eriko Yamada, and Douglas Schneider for their helpful
discussions and technical support.
This work was supported by National Institutes of Health (NIH) grant R01-
A1067031. This project has been funded in part with federal funds from the
National Cancer Institute (NCI), NIH, under contract N01-CO-12400. The content of
this publication does not necessarily refl ect the views or policies of the Department
of Health and Human Services, nor does the mention of trade names, commercial
products, or organizations imply endorsement by the United States Government.
This research was supported in part by the Intramural Research Support Program of
the Center for Cancer Research, NCI, NIH.
The authors have no confl icting fi nancial interests.
Submitted: 6 April 2007
Accepted: 23 October 2007
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with two pairs of specifi c primers for each locus. Internal control primers for
a fragment of 796 bp of the third intron of DRB1 were also included in each
PCR. We amplifi ed 20 − 50 ng DNA in a volume of 20 liters containing 200 M
dNTP, 100 − 500 nM of specifi c primer, 100 nM of internal control primer,
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CD4 expression on HIV-1 – infected CD4 + T cells is reduced. To determine
whether contact was required for NK cell – mediated inhibition of HIV-1
replication, autologous NK cells were physically separated from infected
CD4 + T cells in a transwell experiment, and medium was collected every 3 – 4 d
for p24 Gag quantifi cation by ELISA, as well as for the supernatant add-
For the supernatant add-back experiments, 1 ml of supernatant was
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