BRIEF REPORT: BASIC AND TRANSLATIONAL SCIENCE
NKG2D Expression on HIV-Specific CD8+T cells Is Reduced
in Viremic HIV-1–Infected Patients but Maintained
in HIV Controllers
Camille Lecuroux, PhD,* Asier Saez-Cirion, PhD,† Nicolas Noel, MD,* Lilia Ben-Lamine, PhD,*
Isabelle Girault,* Sophie Caillat-Zucman, PhD,‡ Daniel Scott-Algara, MD, PhD,†
Alain Venet, MD, PhD,* and Olivier Lambotte, MD, PhD*§jj
Abstract: NKG2D mediates an important costimulatory pathway
in CD8+T cells. In HIV infection, the authors found that NKG2D
expression on both total CD8+and HIV-specific CD8+T cells was
significantly lower in viremic patients than in HIV controllers. Anti-
retroviral therapy partially restored NKG2D expression on HIV-
specific CD8+T cells. The authors observed a negative correlation
between the respective expression levels of CD38 and NKG2D
on total CD8+and HIV-specific CD8+T cells. The maintenance
of NKG2D expression on CD8+T cells in HIV controllers may
contribute to better cell function.
Key Words: NKG2D, HIV-specific CD8+T lymphocytes, inflam-
mation, HIV controllers
(J Acquir Immune Defic Syndr 2013;62:17–20)
The CD8+T-cell response has a major role in limiting
HIV replication, as has been demonstrated in simian models,1
primary HIV infection (PHI),2and HIV controllers (HICs, ie,
patients in whom HIV RNA is spontaneously undetect-
able).3,4The mechanisms underlying the strong antiviral
potential and long-term persistence of HIV-specific CD8+T
cells in HICs are not well understood. The NKG2D pathway
could be involved in this phenomenon but remains largely
unexplored in CD8+T cells in HIV-infected patients.
NKG2D is expressed on natural killer (NK) cells and
has a major role in NK-mediated cell lysis.5,6It is also present
on the vast majority of CD8+abT cells.5,7On CD8+T cells,
NKG2D mediates a costimulatory pathway favoring prolifera-
tion, cytotoxicity, and a Th1 antigen–specific cell response.7–11
Conversely, the protein is also involved in CD8+T-cell immu-
noregulation and death. NKG2D ligands (NKG2DLs, such as
the stress-inducible proteins MICA, MICB, and ULBPs) are
induced on activated T cells. The expression of NKG2DLs
may lead to the elimination of activated CD8+T cells by NK
cells, thus limiting their expansion.12,13This could promote
chronic viral infection, as has been recently reported.12More-
over, the release of NKG2DLs downregulates NKG2D expres-
sion on CD8+T cells.14,15
Although it was recently reported that the NKG2D
pathway may have an impact on CD8+T cells in HIV infec-
tion,11the mechanism remains largely unexplored. Thus, we
decided to use flow cytometry to study the expression of
NKG2D and its ligands on T cells, with a focus on HIV-
specific CD8+T cells in different groups of HIV-infected
patients. We hypothesized that decreased expression of
NKG2D and overexpression of NKG2DLs in viremic patients
would lead to the death of HIV-specific T cells. In contrast,
normal NKG2D expression in HICs would optimize cytotox-
icity and cell proliferation.
PATIENTS AND METHODS
Four groups of patients were studied: PHI patients
(n = 11) enrolled in the French ANRS PRIMO cohort,16chron-
ically HIV-1–infected patients with a viral load .10,000 RNA
copies per milliliter (“viremic patients”) (n = 10), patients with
undetectable plasmatic RNA viral load after long-term (.2
years) highly active antiretroviral treatment (HAART) (“trea-
ted patients”) (n = 11), and lastly, HICs (n = 15) enrolled in the
French ANRS HIV Controllers cohort.17The latter patients
had been infected for more than 10 years and had never
received HAART. Ninety percent of their plasma viral RNA
assays were ,400 copies per milliliter.
In addition, 13 healthy donors (HDs) were also studied.
The experimental procedures with human blood were
approved by an independent institutional review board
(Ile de France VII) and were performed according to the
European Union guidelines and the Declaration of Helsinki.
HIV-specific CD8+T cells were identified with soluble
allophycocyanin (APC)-labeled peptide-HLA class 1 multi-
mers (Proimmune, Oxford, United Kingdom) derived from
HIV proteins and then stained with antibodies directed against
NKG2D [coupled to phycoerythrin (R&D Systems Europe,
Received for publication May 28, 2012; accepted September 13, 2012.
From the *INSERM, U1012, Le Kremlin Bicêtre, France; †Institut Pasteur,
Unité de Régulation des Infections Rétrovirales, Paris, France; ‡INSERM
U986, Hôpital St-Vincent de Paul, Paris, France; §Université Paris-Sud,
U1012, Le Kremlin Bicêtre, France; and jjAP-HP, Service de Médecine
Interne et Maladies Infectieuses, Hôpital Bicêtre, Le Kremlin Bicêtre, France.
Supported by grants from Agence Nationale pour la Recherche contre le SIDA
et les Hépatites virales (ANRS), Ensemble contre le SIDA (SIDACTION),
INSERM, and Université Paris XI.
The authors have no conflicts of interest to disclose.
Correspondence to: Olivier Lambotte, MD, PhD, Service de Médecine Interne
et Maladies Infectieuses, CHU Bicêtre, 78 rue du Général Leclerc, F-94275
Le Kremlin Bicêtre, France (e-mail: email@example.com).
Copyright © 2012 by Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr ? Volume 62, Number 1, January 1, 2013www.jaids.com|17
Lille, France)], CD8 [coupled to peridin chlorophyll protein-
cyanine 5.5 (PerCP-Cy5.5)], and CD3 [coupled to APC-H7
(BD Biosciences, San Jose, CA)]. CD38 staining was per-
formed with an antibody coupled to fluorescein isothiocya-
nate (BD Biosciences).
NKG2DLs staining was performed using anti-MICA,
anti-MICB, anti-ULBP1, anti-ULBP2, and anti-ULBP3 anti-
bodies coupled to phycoerythrin (MICA, MICB, ULBP2),
fluorescein isothiocyanate (ULBP1), and APC (ULBP3)
(R&D Systems Europe). The soluble MICA assay was kindly
performed in S. Caillat-Zucman’s laboratory. Statistical anal-
ysis was performed using analysis of variance tests.
NKG2D was strongly expressed on total CD8+T cells
from HDs (as described in the literature5) and HICs (82% 6
15% and 77% 6 20%, respectively) (Fig. 1A). In contrast,
PHI patients showed significantly lower NKG2D expression
on their CD8+T cells (41% 6 22%; P ,0.001 and P , 0.01
when compared with HDs and HICs, respectively). Low
expression was also observed on CD8+T cells from viremic
patients and treated patients (54% 6 18% and 52% 6 16%,
respectively; P , 0.05 vs. HDs for both comparisons).
NKG2D expression was then studied on HIV-specific
CD8+T cells in HIV-infected patients (Fig. 1B). Impor-
tantly, expression was high in HICs (83% 6 14%). In con-
trast, viremic patients and PHI patients, all with high viral
loads, displayed significantly lower expression of NKG2D
on their HIV-specific CD8+T cells, relative to HICs (59% 6
16% for PHI patients and 53% 6 18% for viremic patients;
P , 0.001 vs. HICs for both comparisons). Treated patients
displayed significantly higher expression of NKG2D on their
HIV-specific CD8+T cells (73% 6 16%) than viremic
patients did (P , 0.05). Comparisons of the mean fluores-
cence intensity for NKG2D in each group of patients yielded
similar results (data not shown).
This defect in NKG2D expression may contribute to
impaired function in HIV-specific CD8+T cells because we
observed a positive correlation between NKG2D expression
on one hand and the percentages of both IL-2+and CFSElow
CD8+T cells on the other hand (P = 0.002, r = 0.49, and
P = 0.031, r = 0.38, respectively).
Our results suggest that HIV infection lowers the
expression of NKG2D on total CD8+and HIV-specific
CD8+T cells. We then investigated several possible mecha-
nistic explanations for the much lower levels of NKG2D on
CD8+T cells from viremic patients.
First, reports in many settings have demonstrated that
soluble forms of MHC class I chain-related (MIC) molecules
induce NKG2D downmodulation in NK and T cells.14,15We
therefore tested for the presence of soluble MICA in the
patients’ plasma; however, low levels were found in all the
groups (data not shown).
Second, activated CD8+T cells may express NKG2DLs,
which would lead to their killing by NK cells.12,13We and
others have reported that viremic subjects have higher activated
CD8+T-cell counts than HICs and HAART-treated patients.4,18
Thus, we hypothesized that there could be a negative correla-
tion between NKG2D expression and CD38 expression and
a positive correlation between NKG2DLs expression and
CD38 expression in CD8+T cells from untreated patients.
Indeed, we observed a negative correlation between the respec-
tive expression levels of CD38 and NKG2D on total CD8+T
cells (Fig. 2A, P = 0.0045, r = 20.35) and HIV-specific CD8+
T cells (Fig. 2B, P = 0.0003 and r = 20.46). In contrast, we did
not detect any significant ex vivo expression of MICA, MICB,
or ULBP1, ULBP2, and ULBP3 on CD8+T cells (data not
shown) and therefore no correlation with activation of expression.
This finding suggests either that there is a defect in NKG2DLs
FIGURE 1. A, NKG2D expression on total CD8+T cells [***P , 0.001, **P , 0.01, *P , 0.05 in an analysis of variance (ANOVA)].
B, NKG2D expression on HIV-specific CD8+T cells from HIV-infected patients (***P , 0.001, **P , 0.01, *P , 0.05 in an ANOVA).
Lecuroux et al
J Acquir Immune Defic Syndr ? Volume 62, Number 1, January 1, 2013
? 2012 Lippincott Williams & Wilkins
expression on CD8+T cells in the setting of HIV infection or that
NKG2DL-expressing activated CD8+T lymphocytes are rapidly
killed by NK cells.
To test the first hypothesis, we investigated the expression
of NKG2DLs on CD8+T cells after strong mitogenic activation
(with a combination of anti-CD2 and anti-CD28 antibodies) in
the different groups of patients. Each ligand tested was signif-
icantly and similarly induced in vitro on activated CD8+T cells
from each group (data not shown). Next, we looked at whether
the level of NKG2D expression on NK cells could account for
the killing of NKG2DL+CD8+T cells. We found that HDs and
viremic patients did not differ significantly in terms of NKG2D
expression on their NK cells (74% 6 6% and 69% 6 7%,
respectively), as previously reported.19Unexpectedly, NKG2D
expression was significantly lower on NK cells from HICs
when compared with those from viremic patients or HDs
(13% 6 3%, P , 0.0001 for both comparisons).
Our results suggest that HIV replication lowers the
expression of NKG2D on total CD8+and HIV-specific CD8+
T cells. This costimulatory defect may contribute to impaired
function in anti-HIV CD8+T cells. In contrast, NKG2D expres-
sion is maintained on total CD8+and HIV-specific CD8+T cells
The level of immune activation may play a role in the
regulation of NKG2D expression on CD8+T cells because we
observed a negative correlation between the respective expres-
sion levels of CD38 and NKG2D on total CD8+T cells and
HIV-specific CD8+T cells. In the context of antiretroviral
therapy, reduced activation might allow the reconstitution of
a pool of HIV-specific NKG2D+CD8+T cells. Interestingly,
normal expression of NKG2D on HIV-specific CD8+T cells in
HICs may be favored by the lower level of immune activation
seen in these patients when compared with viremic individu-
als.18Given that the MICA and MICB proteins are highly
polymorphic,20rare alleles associated with reduced cell mem-
brane expression of these proteins could be overrepresented in
HICs (thus reducing CD8+T-cell killing by NKG2D+NK
cells). Interestingly, a genome-wide association study of the
HIC cohort showed that most of the tag single-nucleotide poly-
morphisms associated with low viral replication were located
in the region ranging from HLA-Cw to MICB.21Low in vivo
induction of MICA or MICB on CD8+T cells by physiological
stimuli could lead to the greater maintenance of HIV-specific
NKG2D+CD8+T-cell function in HICs. This point deserves
However, we also showed that there was neither an
overall nor a selective impairment of the expression of the
various NKG2DLs on CD8+T cells after in vitro cell activa-
tion. Our results thus suggest that NKG2DL+CD8+T cells
are not detected ex vivo because they are killed in vivo by
NKG2D+NK cells. After looking at NKG2D expression on
NK cells in viremic patients and HICs, our results are in
accordance with the literature: in viremic patients, NKG2D
expression is maintained on NK cells (in contrast to other
expression of NKG2D on NK cells in HICs has not been
previously described and remains puzzling. Other peculiar
phenotypic features in controllers have suggested the specific
regulation of activating NK cell receptors in these patients.22
Low expression of NKG2D on NK cells might also facilitate
the survival of HIV-specific CD8+T cells in HICs.
This extensive study is the first to have described
NKG2D expression on total CD8+and HIV-specific CD8+T
cells in different groups of HIV-infected patients. We suggest
that the maintenance of NKG2D expression on CD8+T cell in
HICs may help optimize CD8+T-cell function and the anti-
viral immune response in these patients, as recently sug-
gested.11Therapeutic strategies based on reduced immune
activation should be also beneficial in the maintenance of this
cell activation pathway.
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FIGURE 2. A, Negative correlation
NKG2D+CD8+T cells and activated
CD38+CD8+T cells. B, Negative
correlation between the percentages
of HIV-specific NKG2D+CD8+T cells
and HIV-specific activated CD38+
J Acquir Immune Defic Syndr ? Volume 62, Number 1, January 1, 2013NKG2D Expression on CD8+T cells in HIV Infection
? 2012 Lippincott Williams & Wilkins
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? 2012 Lippincott Williams & Wilkins