A Molecular Basis for the Control
of Preimmune Escape Variants
by HIV-Specific CD8+T Cells
Kristin Ladell,1,11Masao Hashimoto,2,11Maria Candela Iglesias,3,11Pascal G. Wilmann,4,11James E. McLaren,1
Ste ´phanie Gras,4Takayuki Chikata,2Nozomi Kuse,2Sole `ne Fastenackels,3Emma Gostick,1John S. Bridgeman,1
Vanessa Venturi,5Zaı ¨na Aı ¨t Arkoub,6Henri Agut,6David J. van Bockel,7Jorge R. Almeida,3,8Daniel C. Douek,8
Laurence Meyer,9Alain Venet,9Masafumi Takiguchi,2,12Jamie Rossjohn,1,4,12David A. Price,1,8,12
and Victor Appay3,10,12,*
1Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK
2Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
3INSERM UMR S 945, Infections and Immunity, Universite ´ Pierre et Marie Curie-Paris6, Ho ˆpital Pitie ´-Salpe ˆtrie `re, 75013 Paris, France
4Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria 3800, Australia
5Computational Biology Group, Centre for Vascular Research, University of New South Wales, Kensington, Sydney, NSW 2052, Australia
6Virology Laboratory, ER1 DETIV UPMC, Universite ´ Pierre et Marie Curie-Paris6, Ho ˆpital Pitie ´-Salpe ˆtrie `re, 75013 Paris, France
7St Vincent’s Centre for Applied Medical Research and University of New South Wales, Darlinghurst, Sydney, NSW 2010, Australia
8Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
9INSERM, Universite ´ Paris-Sud, Ho ˆpital du Kremlin-Bice ˆtre, 94275 Le Kremlin-Bice ˆtre, France
10AP-HP, Groupe Hospitalier Pitie ´-Salpe ˆtrie `re, Laboratoire d’Immunologie Cellulaire et Tissulaire, 75013 Paris, France
11These authors contributed equally to this work
12These authors contributed equally to this work
The capacity of the immune system to adapt to
rapidly evolving viruses is a primary feature of
effective immunity, yet its molecular basis is
unclear. Here, we investigated protective HIV-1-
specific CD8+T cell responses directed against the
immunodominant p24 Gag-derived epitope KK10
(KRWIILGLNK263-272) presented by human leukocyte
antigen (HLA)-B*2705. We found that cross-reactive
CD8+T cell clonotypes were mobilized to counter
the rapid emergence of HIV-1 variants that can
directly affect T cell receptor (TCR) recognition.
These newly recruited clonotypes expressed TCRs
that engaged wild-type and mutant KK10 antigens
with similar affinities and almost identical docking
modes, thereby accounting for their antiviral efficacy
in HLA-B*2705+individuals. A protective CD8+T cell
repertoire therefore encompasses the capacity to
control TCR-accessible mutations, ultimately driving
the development of more complex viral escape vari-
ants that disrupt antigen presentation.
Adaptive CD8+T cell immunity is critical for protection against
viruses and other intracellular pathogens. At the molecular level,
this host-pathogen conflict centers on T cell receptor (TCR)
engagement of major histocompatibility complex class I
(MHC-I) molecules bearing peptide fragments derived from the
intracellular invader. The ab TCR repertoire encompasses
a phenomenal level of diversity, which is generated by somatic
recombination of variable (V), diversity (D), and joining (J)
gene segments, junctional modifications, and differential
pairing of a and b chains. Theoretically, between 1015and 1020
different TCRs can be generated by this process (Davis and
Bjorkman, 1988; Lieber, 1991). However, because of size
toire estimated to contain around 2.5 3 107TCRs (Arstila et al.,
1999). The diversity of the peripheral TCR repertoire has
profound implications for effective immune coverage (Nikolich-
Zugich et al., 2004).
Rapidly evolving pathogens capable of intrahost evolution
during the course of infection must contest with the array of
TCRs available for deployment. This sets the stage for a ‘‘molec-
ular arms race’’ between the pathogen and the host. Immune
escapebymutationoftargeted CD8+Tcellepitopes isacardinal
feature of HIV-1 infection and represents a key obstacle to the
successful development of an AIDS vaccine (Goulder and Wat-
kins, 2004). Indeed, the intense antiviral pressure exerted by
CD8+Tcellresponses drivestherapidselection ofescapemuta-
tions from the earliest stages of infection (Goonetilleke et al.,
2009). Eventually, the frequency of such mutations correlates
with the prevalence of the restricting human leukocyte antigen
(HLA) class I allele across the population as a whole (Dong
et al., 2011; Kawashima et al., 2009; Moore et al., 2002). None-
theless, certain HLA class I alleles, including HLA-B*27, can
confer relative protection from disease progression (Kaslow
Immunity 38, 425–436, March 21, 2013 ª2013 Elsevier Inc. 425
the immunodominant KK10 epitope (KRWIILGLNK263-272) in
p24 Gag is almost invariably targeted by CD8+T cells (Altfeld
et al., 2006; Scherer et al., 2004). These KK10-specific CD8+
T cells unleash potent effector functions (Almeida et al., 2007;
Berger et al., 2011), and the conservation of this response is
thought to account for the benefits conferred by HLA-B*27
A number of mutations in the KK10 epitope have been
reported in HIV-1-infected patients. The commonly observed
Leu268Met mutation was initially considered to be a compensa-
tory change required for the later appearance of the Arg264Lys
mutation, which isassociated with increased viralloads and clin-
ical progression (Ammaranond et al., 2011; Feeney et al., 2004;
Goulder et al., 1997; Kelleher et al., 2001). However, we recently
showed that the Leu268Met mutation enables HIV-1 to evade
defined by the expression of public TRBV4-3 TRBJ1-3 TCRs,
which can be shared between individuals responding to the
KK10 epitope (Iglesias et al., 2011). Nonetheless, control of viral
replication is usually preserved despite this mutation. In HIV-1-
infected patients presenting primarily a Leu268Met virus, the
KK10-specific CD8+T cell response seems to be more reactive
against the Leu268Met variant compared to the wild-type (WT)
epitope; the converse applies in patients presenting a predomi-
nant WT virus (Iglesias et al., 2011; Lichterfeld et al., 2007;
Streeck et al., 2008). These observations suggest that the
immune response can adapt to mutations within the KK10
peptide that remain accessible to the TCR repertoire. However,
the precise mechanism responsible for the maintenance of
immune control in this situation remains unclear. Here, we
show that the immune system can counter the emergence of
Leu268Met variants through the mobilization of newly generated
cross-reactive KK10-specific CD8+T cells, in particular those
bearing TRBV6-5 TRBJ1-1 TCRs. These clonotypes were able
to control both WT and Leu268Met viruses and retained potent
suppressive capacity unless the virus succeeded in acquiring
mutations, such as Arg264Lys, that negatively impact epitope
Emergence of Cross-Reactive WT and Leu268Met
KK10-Specific CD8+T Cells during Primary HIV-1
To understand how KK10-specific CD8+T cell populations
adjust to the early emergence of the Leu268Met mutant and
maintain control of viral replication, we examined the fine
specificity of responses to this epitope in HLA-B*2705+patients
with primary HIV-1 infection. For this purpose, WT, Leu268Met,
or dually reactive KK10-specific CD8+T cells were identified
directly ex vivo by flow cytometry with fluorescent HLA-
B*2705-peptide tetramers (Figure 1A). During early infection
(i.e., within 3 months of viral transmission), robust CD8+T cell
responses against WT, but not Leu268Met, KK10 could be de-
tected in three patients (Figures 1A and 1B). These CD8+T cell
populations engaged WT KK10 antigen (Ag) with high avidity,
as revealed by staining with the corresponding CD8-null
tetramer. Previous studies have shown that this physical param-
eter correlates with potent HIV-suppressive capacity (Almeida
et al., 2007, 2009; Berger et al., 2011). Subsequently, we
observed CD8+T cell expansions with reactivity against
the Leu268Met variant (Figures 1A and 1B). These distinct
populations exhibited high avidities for the cognate Ag in
two patients (PrInf A and B) with stable control of viral replication
in the absence of antiretroviral therapy. In contrast, lower-
avidity Leu268Met KK10-specific CD8+T cells were present in
patient PrInf C, who initiated antiretroviral therapy 18 months
after infection as a result of progressively increasing viral loads
Extensive viral sequencing, from both plasma and peripheral
blood mononuclear cells, was conducted to inform the observed
patterns of KK10 reactivity (Figure 1C and Table S1 available
online). In PrInf C, expansion of the Leu268Met KK10-specific
CD8+T cell population coincided with the emergence of
detectable Leu268Met variant virus at month 18. Indeed, the
Leu268Met sequence was dominant at the cell-associated
DNA level, indicating that the corresponding variant virus was
poorly controlled in this patient. In patients PrInf A and B, how-
ever, the high-avidity Leu268Met KK10-specific CD8+T cell
tion. Indeed, the Leu268Met sequence was detected in the cell-
associated DNA from PrInf A only at a very late time point, after
the initiation of antiretroviral therapy. Collectively, these data
indicate that the immune system can generate high-avidity
cross-reactive WT and Leu268Met KK10-specific CD8+T cells
soon after primary HIV-1 infection, even with frequencies of
Leu268Met variant virus that lie below the limit of detection
with conventional sequencing methodologies.
High-Avidity WT and Leu268Met KK10-Specific CD8+
T Cells Incorporate TRBV6-5 TRBJ1-1 Motif-Bearing
To characterize the molecular basis for WT and Leu268Met
cross-reactivity at the level of TCR usage, we performed
ex vivo analyses of TRB gene expression in both WT and dually
reactive WT and Leu268Met KK10-specific CD8+T cell popula-
tions (Figure 2). CD8+T cell populations specific for the WT
epitope were characterized by the turnover of recurrent clono-
types, several of which used the TRBV4-3 gene as described
previously (Iglesias et al., 2011). In contrast, the WT and
Leu268Met KK10-specific CD8+T cell populations comprised
entirely distinct clonotypes, suggesting de novo mobilization
from the naive pool. The high-avidity WT and Leu268Met
KK10-specific CD8+T cells observed in patients PrInf A and
PrInf B showed particular enrichments for TRBV6-5 TRBJ1-1
clonotypes. Furthermore, TRBV6-5 TRBJ1-1 clonotypes were
detected in both WT and Leu268Met KK10-specific CD8+
T cell populations in two untreated HLA-B*2705+patients during
the chronic phase of HIV infection (ChInf D and E; Figure S1A).
The CDR3b sequence differed by a maximum of three amino
acids between these TCRs (Figure 1D), indicating that a
TRBV6-5 TRBJ1-1 motif (CASRXGXGXTEAF) can underpin WT
and Leu268Met cross-reactivity. Of note, a similar CDR3b
sequence was recently detected in an HIV-1-infected patient
with elite control of viral replication (Chen et al., 2012). Molecular
analysis of TRA gene expression revealed that the TRBV6-5
TRBJ1-1 clonotypes were accompanied by TRAV14 TRAJ21
transcripts in the respective CD8+T cell populations from PrInf
Coadaptation of HIV and CD8+T Cell Immunity
426 Immunity 38, 425–436, March 21, 2013 ª2013 Elsevier Inc.
assays with HLA-B*2705+EBV-transformed B cell targets as described previ-
ously (Almeida et al., 2007, 2009). For functional profiling, CD8+T cell clones
were incubated for 1 hr at 37?C in the presence of aCD107a mAb and HLA-
HIVNL4-3virus; monensin (2.5 mg/ml; Sigma-Aldrich) and brefeldin A (5 mg/ml;
Sigma-Aldrich) were added for a further 5 hr. Staining for intracellular markers
and data analysis were performed as described previously (Almeida et al.,
2009). For HIV suppression assays, primary HLA-B*2705+CD4+T cells were
purified from PBMCs by positive magnetic bead selection (Miltenyi Biotec),
stimulated for 2 days with PHA (1 mg/ml), and cultured with 100 U/ml rhIL-2.
Seven days later, 105cells/well were infected with titrated levels of viruses by
spinoculation (Iglesias et al., 2011) and mixed with CD8+T cell clones at
different CD8+/CD4+ratios. Cells were harvested at day 3 postinfection, then
stained for both CD4 and intracellular p24 to evaluate the elimination of HIV-
as follows: from BD Biosciences, aCD4-APCCy7, aCD107a-Cy5PE, aIL-2-
APC, aIFNg-Alexa700, and aTNFa-PECy7; from Caltag Laboratories, aCD8-
Alexa405; from R&D Systems, aMIP-1b-FITC; and from Beckman Coulter,
ap24-PE. The viability dye ViViD (Life Technologies) was used to eliminate
dead cells from flow cytometric analyses.
Protein Production and Crystallography
Soluble recombinant proteins for structural and binding studies were pro-
duced as described previously with minor modifications (Gras et al., 2009;
Iglesias et al., 2011; Price et al., 2005). Crystals of the C12C TCR in complex
with WT and Leu268Met HLA-B*2705-KK10 were grown by the hanging-
drop, vapor-diffusion method at 20?C with a protein reservoir drop ratio of
1:1 and a concentration of 5 mg/ml in 10 mM Tris (pH 8), 150 mM NaCl. Crys-
talsgrewin24%PEG 3350and 0.3MNa2SO4.CrystalsoftheWT andLeu268-
Met HLA-B*2705-KK10 complexes were grown by the same technique in
a mother liquor containing 16% PEG 8000 and 0.1 M MES (pH 7.0). Data
collection, processing, structure determination, refinement, and validation
were conducted with standard crystallography software. Details are provided
in Supplemental Information online.
p values less than 0.05 were considered significant.
The atomic coordinates and structure factors were deposited in the Protein
Data Bank with the following accession codes: 4G8G for the C12C TCR in
complex with WT HLA-B*2705-KK10, 4G9F for the C12C TCR in complex
with Leu268Met HLA-B*2705-KK10, 4G9D for WT HLA B*2705-KK10, and
4G8I for Leu268Met HLA B*2705-KK10.
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and two tables and can be found with this article online at http://
We thank all study participants, the French Agence Nationale de la Recherche
sur le SIDA (ANRS) Cohorts ALT and PRIMO groups, and the staff at the MX2
infected cells at the Pitie ´-Salpe ˆtrie `re Flow Cytometry Platform. This work was
supported by the ANR (project ANR-09-JCJC-0114-01), Sidaction, the French
ANRS, the National Institutes of Health via the Intramural Program of the
Vaccine Research Center (National Institute of Allergy and Infectious
Diseases), the Australian Research Council (ARC), the Australian National
Health and Medical Research Council (NHMRC), the UK Biotechnology and
Biological Sciences Research Council (grant BB/H001085/1), the Japanese
Ministry of Health (grant 18390141), and the Global COE program (‘‘Global
Education and Research Center Aiming at the Control of AIDS’’) of the Japa-
nese Ministry of Education, Science, Sports, and Culture. M.C.I. is supported
by a Sidaction Fellowship. V.V. and S.G. are ARC Future Fellows. P.G.W. is an
NHMRC CJ Martin Fellow. J.R.is anNHMRCAustralia Fellow. D.A.P. isaWell-
come Trust Senior Investigator.
Received: March 24, 2012
Accepted: November 5, 2012
Published: March 21, 2013
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