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Laboratoire d’Immunologie des Pathologies infectieuses et tumorales, Institut National de la Santé et de la Recherche Médicale, Unité 445, Institut Cochin de Génétique Moléculaire, Université René Descartes, Paris, France; and
Laboratoire d’Immunologie et Chimie Thérapeutiques, Centre National de la Recherche Scientifique, Unité Propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In MHC class I peptide presentation, the nature of the HLA alleles that select and bind peptides is important because only HLA-peptide complexes of sufficient binding affinity and stability are loaded and presented to TCRs (7, 8, 9). Degradation of Ag, binding of liberated peptides to MHC molecules, and epitope recognition by T cells must all be assessed to provide insight into the dynamic interplay that exists between Ag and T cell response, as suggested by Koup (10).
To study the antigenic potential of a protein and to identify regions
that could provide multiple T epitopes, we selected the HIV-1 Nef
protein. Its relatively small size (
200 amino acids), the knowledge
of its structure in solution (11), and the high frequency
of CTL responses directed against this protein in HIV-1-infected
individuals are the factors that governed this choice.
In the present report, we analyze the distribution of HLA-specific anchor residues, the HLA-binding capacity of relevant peptides, and the frequency of epitope presentation by each of six common HLA alleles. To examine Nef processing, we investigated the proteasome cleavage of the epitope-rich region 66–100. Relationships between Nef structure, processing, and CTL epitope recognition were then sought.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides. Synthetic peptides of 8–11 residues containing putative anchor-binding motifs for HLA-A2, -A3, -A24, -B7, -B8, and -B35 were selected from the Nef HIV-1/Bru sequence. They were synthesized by Chiron Mimotopes (Victoria, Australia) or Neosystem (Strasbourg, France). They were supplied by Agence Nationale de la Recherche sur le SIDA. Lyophilized peptides were dissolved in DMSO and diluted to 1 mg/ml in water (final concentration of DMSO, 10%), aliquoted, and stored at -20°C.
HLA molecules.
HLA molecules were purified from EBV-transformed B cell lines. After
cell lysis in PBS with 1% Nonidet P-40 and protease inhibitors, HLA
molecules were retained on affinity columns using anti-HLA Igs,
eluted, and then frozen at -80°C. HLA molecules were denatured in
PBS containing 12.5 mM NaOH (pH 11.7) and 1.5 M urea for 1 h at
4°C. HLA H chains and 
2-microglobulin
(2m)3
were separated from endogenous peptides on a Sephadex G25 column (PD10;
Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in PBS
containing 0.05% Tween 20, 2 mM EDTA, and 0.1% Nonidet P-40, as
previously reported (12). Then 2 µg/ml exogenous
2m (Sigma) and 6 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(Sigma) were added just before addition of exogenous
peptide.
HLA-peptide binding.
Aliquots of HLA H chains (1 µg in 50 µl) were incubated with
different concentrations (10-4 and
10-6 M) of exogenous peptides in Eppendorf
microtubes (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) for
1 h at room temperature and then for 24 h at 4°C.
Reassembled HLA molecules were further incubated for 90 min at 37°C
in wells of microtiter plates coated with anti-HLA Abs
(13). Correctly folded HLA complexes were revealed with
anti-2m Ig coupled to alkaline
phosphatase, with 4-methyl-umbelliferyl phosphate (M-8883; Sigma, St.
Louis, MO) as substrate. Fluorescence generated was measured at 360/460
nm in a Microfluor reader (Victor 1420; Wallac, Turku, Finland).
HIV-1-seropositive donors and detection of T cells recognizing Nef peptides
Lymphocytes donors. PBMC from 76 HIV-1-seropositive individuals were isolated by density gradient centrifugation (separation medium; Flow, Irvine, U.K.) and used after freezing and thawing. Cohorts were established with the approval of the local ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale of Cochin hospital), and all participants gave their written informed consent for the constitution of cell banks.
Enzyme-linked immunospot (ELISPOT) assay.
The IFN-
ELISPOT assay was adapted from Scheibenbogen et al. and
described by Dalod et al. (14). Ninety-six-well
nitrocellulose plates (Millipore, Bedford, MA) were coated with 2
µg/ml mouse anti-human IFN- mAb (number 1598-00; Genzyme,
Rüsselheim, Germany). PBMCs, either freshly isolated or thawed,
were cultured overnight in complete medium (RPMI 1640 supplemented with
Glutamax, nonessential amino acids, sodium pyruvate (1 mM), HEPES
buffer (10 mM), penicillin (100 U/ml), streptomycin (100 mg/ml) (Life
Technologies, Paisley, U.K.), and 10% FCS (PAN Biotech, Aidenbach,
Germany)) and plated in triplicate at serial dilutions (3 x
105–104 cells/well).
Appropriate stimuli were then added, and the plates were incubated for
20 h at 37°C in 5% CO2. After washing,
the cells were incubated with 100 µl rabbit polyclonal anti-human
IFN- Ab diluted 1:250 (IP500; Genzyme), then with a biotinylated
anti-rabbit Ig G diluted 1:500 (Boehringer Mannheim, Mannheim,
Germany), and finally with alkaline phosphatase-labeled extravidin
(Sigma). Spots were developed by adding chromogenic alkaline
phosphatase substrate (Bio-Rad, Hercules, CA), and colored spots were
counted in a stereomicroscope. A result was considered to be
significant when the numbers of spots were at least twice the
background value (value given by negative peptides) and were
proportional to the numbers of plated cells. Frequencies of IFN-
spot-forming cells (SFC) were calculated. Positive controls for IFN
detection consisted of six wells containing 300–1000 cells stimulated
with 50 ng/ml PMA and 500 ng/ml ionomycin. This strong mitogenic
stimulus verified that freezing and thawing did not introduce
artifacts, and it constituted an indirect check of overall T cell
viability. Negative controls for immune recognition consisted of
epitopes derived from various viruses (for example, peptide
Tax11–19 from human T cell lymphotropic
virus-1, which associates with HLA-A2); they never elicited a
significant response compared with PBMCs incubated in medium alone.
Positive controls for immune recognition were epitopes of EBV or
influenza virus. By using purified T cell subsets, we verified that
IFN- was secreted by CD8+ cells.
Degradation of large Nef peptide 66–100
Proteasome digestion. Peptide 66–100 from Nef HIV-1/Bru had the following sequence: VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGL. It was synthesized by Neosystem. The peptide (150 µg) was incubated at 37°C with 30 µg of 20S proteasome (539150; Calbiochem, La Jolla, CA) in 200 µl assay buffer containing 20 mM Tris-HCl (pH 8) and 0.5 mM EDTA for times varying from 4 to 24 h. Cleavage products were separated by reversed phase HPLC (Perkin-Elmer, Norwalk, CT) on a C18 column (Nucleosil; 10 µm, 250 x 4.0 mm; Macherey-Nagel, Hoerdt, France) using a linear gradient of 0.1% trifluoroacetic acid in water and 0.08% trifluoroacetic acid in acetonitrile, 1% of the latter in 5 min, 1–10% in 5 min, 10–35% in 50 min, and 35–60% in 10 min at 0.8 ml/min flow rate. Chromatogram was recorded at 214 nm (759 Å; Applied Biosystems, Roissy, France). Major peaks (absorbance above 0.15 absorbance unit (AU) were collected and lyophilized.
Detection of epitopes in proteasome digests.
Thawed PBMCs of patient Z037 were cultured overnight and plated in a
96-well nitrocellulose plate at 4 x 105
cells/well in 100 µl of complete medium. Lyophilized fractions
were dissolved in complete medium, and 100 µl/well were added on
cells. ELISPOT IFN- were revealed as described before after
overnight incubation.
Mass spectrometry analysis and Edman’s sequencing.
Mass analysis was performed on a linear matrix-assisted laser
desorption ionization time-of-flight Bruker (Billerica, MA) instrument
using 
-cyano-4-hydroxycinnamic acid as matrix. Peptides were
sequenced by automated Edman degradation using an Applied Biosystems
Procise CLC protein sequencer equipped with a PTH ABI 140D analyzer
(Applied Biosystems, Foster City, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight to eleven-mer Nef peptides with binding motifs specific for
six common HLA molecules, as defined by Sidney and Sette (15, 16), are shown in Fig. 1
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Twenty-two of these peptides were for HLA-A2 (L, I, M, V, or A at
position 2 and V, L, or A at the C-terminal), 18 peptides for HLA-A3
with a motif requiring the presence of positively charged residues (L,
V, A, I, or T at position 2 and K, R, or Y at the C-terminal), 13
peptides for HLA-A24 (Y or F at position 2 and L, F, Y, or I at the
C-terminal), 23 peptides for HLA-B7/B35 (P or S at position 2 and a
hydrophobic residue at the C-terminal), and 5 peptides with
HLA-B8-specific motif (basic residue R or K, or Q at positions 3 and 5,
and hydrophobic residue at the C-terminal). Thus, we found 81 motifs,
and we defined 73 peptides containing one or two HLA-binding motifs
(Fig. 1). Nef regions containing multiple motifs are, in decreasing
order: 126–153 (15 motifs in 28 residues), 65–112 (25 motifs in 48
residues), 180–204 (13 motifs in 25 residues), and 9–23 (7 motifs in
15 residues). If we consider the distribution of various motifs,
HLA-A2, -A3, or -B7/35-specific motifs are observed in all regions,
whereas HLA-A24-specific motifs are mainly located in the central
region, which contains a high frequency of hydrophobic and aromatic
residues.
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We then tested the binding of all these peptides to
the six HLA molecules. We used purified HLA H chains and synthetic
peptides as described in Materials and Methods. For each HLA
molecule, we tested a reference epitope that yielded 100% binding (see
legend of Fig. 2). The most significant
results were obtained at a peptide concentration of
10-6 M. We defined as high binders
the peptides yielding 50–100% binding and as moderate binders the
peptides yielding 20–50% binding. We also considered as low binders
the peptides yielding <20% binding at 10-6 M
and >20% at 10-4 M.
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Recognition of Nef peptides by T cells from HIV-1 infected patients
Nineteen synthetic peptides from binder-rich regions 66–100 and
126–146 were tested for their capacity to stimulate PBMCs from
HIV-1-infected patients ex vivo. The induction of IFN- secretion was
revealed by ELISPOT. Only significant positive results (more than twice
the background value for each individual) were retained. An epitope may
be defined by peptide recognition by PBMCs from only one
patient. Seventeen peptides were positive. Frequencies of T cell
recognition are given in Table I. A high
T cell recognition frequency (T cell recognition observed in at least
50% of the donors tested) was obtained with eight peptide-HLA
complexes yielding high (three of eight), moderate (three of eight), or
low (two of eight) HLA binding, respectively. Moderate T cell
recognition frequency (25 to <50%) was observed in six peptide-HLA
complexes yielding high (one of six), moderate (three of six), or low
(two of six) HLA binding. Low T cell recognition frequency was observed
in eight peptide-HLA complexes yielding high (two of eight), moderate
(five of eight), or low (one of eight) HLA binding. The frequency of T
cell recognition was not directly related to the strength of HLA
binding, which shows the influence of other factors, notably those
related to Ag processing, epitope variations, or available repertoire.
The peptides most frequently recognized were, in decreasing order,
90–97, 135–143, 71–81, 77–85, 90–100, 73–82, and 128–137. We
determined six new epitopes: 71–79 and 71–81 presented by HLA-B7/B35,
83–91 by HLA-A2, 126–135 by HLA-A24, 130–139 by HLA-B35, and
136–144 by HLA-A3.
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The large number of epitopes represented in Fig. 3 and the high frequency of T cell
recognition of some of them possibly result from an efficient
processing of the Nef regions 66–100 and 126–146 with liberation of
adequate peptides. As the processing of the second Nef region has
already been studied by Lucchiari-Hartz et al. (17), our
study focused on in vitro cleavage of the first region,
Nef66–100. A recombinant 20S proteasome with
mainly chymotrypsin-like activity was used. Degradation was controlled
by reversed phase HPLC chromatography after various incubation times at
37°C; 24 h incubation was the optimum time (data not shown). The
major peak of the HPLC chromatogram corresponding to
Nef66–100 (eluted after 67 min) greatly
decreased after 24 h incubation, whereas numerous other peaks
appeared between 30 and 55 min. Fractions corresponding to peaks
with absorbance higher than 0.15 AU were collected. A partial HPLC
chromatogram of the peptides that separated at 40–70 min is
represented in Fig. 4A. We
chose to study the generation of epitope
Nef71–81 because of its central position in
Nef66–100 sequence and the presence of flanking
sequences that may influence processing. As shown in Fig. 4B, HIV-1-seropositive patient Z037 presented a very strong
response against this epitope (2800 SFC/106
PBMC). T cells of this patient were used to screen the presence of this
epitope in HPLC fractions. Peptides eluted before 40 min were not
recognized (data not shown). As shown in Fig. 4C, fractions
C and D induced strong reactivity (>1000 SFC/106
PBMC). Analysis of peptide contents of these fractions by mass
spectrometry and Edman sequencing revealed the presence of peptide
69–81 in C and 68–81 in D (Table II).
These two species are NH2-extended forms of the
Nef71–81 epitope. Fraction A was not recognized
by Z037 T cells, and only Nef72–81 was
characterized but was too short for inducing a stimulation. Fractions B
and E were recognized by a smaller number of effector T cells (500
SFC/106 PBMC), and analysis of their content
revealed that major species were Nef88–100
(fraction B) and Nef84–100 (fraction E). T cells
from patient Z037 recognized Nef90–100 with an
intensity of 570 SFC/106 PBMCs, and they probably
also recognized extended forms of this epitope
(Nef88–100 and
Nef84–100). However, demonstration of cleavage
by 20S proteasome at position 100 would require the use of another
COOH-terminally extended peptide. There was a relationship between the
number of T effector cells recognizing the optimal peptide
Nef71–81 (2800 SFC/106
pulsed PBMCs) and the number of T cells recognizing the
NH2-extended forms
Nef68–81 and Nef69–81 in
fractions C and D (>1000 SFC/106 pulsed
PBMCs). Similarly, peptide Nef90–100
activated 570 T effector cells, and fractions B and E, containing
NH2-extended forms of this epitope, activated
500 effectors. In summary, we have characterized the efficient
generation by 20S proteasome of different forms of
Nef71–81, which is an epitope having a high
frequency of T cell recognition in HIV-infected patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In Nef,
such peptides can be identified along the whole sequence, but notably
in the central region 66–146(66–146), which is characterized by numerous
hydrophobic residues, proline, and some basic residues. Peptides that
truly bound to these HLA molecules were remarkably concentrated in
regions 66–100 and 126–146 (Fig. 2).
We chose to study further the presentation of peptides from these two
regions and their T cell recognition. A high proportion of peptides
containing HLA-A3- and HLA-B7/B35-specific motifs were good binders,
which is consistent with the high frequency of T cell recognition of
several epitopes presented by these HLA molecules. For HLA-A2, a lower
binding is also consistent with the lower frequency of Nef
epitopes identified (Table I). Among the peptides that can bind to the
6 HLA molecules tested (Fig. 2), 19 were tested against T cells of 76
HIV positive patients; 17 of these induced stimulation. The frequency
of T recognition was especially high for epitopes 90–97 (presented by
HLA-B8), 135–143 (B35), 71–81 (B7 and B35), 77–85 (B7), 90–100
(A2), 73–82 (A3), and 128–137 (B7). This result suggests that few
recognition-inhibiting variations occur in these Nef segments. It may
also reveal that cellular processing liberates high quantities of these
peptides.
Although several enzymatic systems are involved in protein digestion,
proteasomes probably generate the majority of cytosolic peptides
(18). Niedermann et al. showed that processing of
polypeptides by proteasomes is conserved in evolution
(19). In APCs, IFN- induction alters proteasomal
proteolysis in such a way that the generation of peptides with
structural features of MHC class I ligands might be optimized (4, 5). Cleavage after basic residues is possible after this IFN-
treatment, which enhances trypsin-like activity, but the majority of
changes may be quantitative rather than qualitative. In the present
report, by studying digestion of peptide
Nef66–100 by a 20S proteasome having a
chymotrypsin-like activity, we identified the efficient production of
NH2-extended forms of epitope 71–81. When T
cells of HIV-1-infected individuals were tested, peptide 71–81 was
recognized by 75% of HLA-B7-typed donors and by 50% of HLA-B35-typed
donors (Table I). Mass spectrometry and Edman’s sequencing showed that
two NH2-extended forms,
Nef68–81 and Nef69–81,
were efficiently produced by 20S proteasome (Table II). In the case of
epitope Nef71–81, HLA-B7 and -B35 bindings were
low and moderate, respectively. Dominance of T cell response could be
explained by a high concentration of liberated peptides and could
therefore be very dependent on proteasome cleavage. It is interesting
to note that another epitope, Nef74–81, was
recognized by 31% of HLA-B35-typed patients, and this epitope could
also have Nef68–81 and
Nef69–81 as precursors. However, HLA-B7-typed
patients did not recognize Nef74–81 because of
its failure to bind to this HLA allele. Of the other peptides from the
same region, peptide 68–76 bound very efficiently to HLA-B7, but its
frequency of T recognition was low, which could be consistent with a
very low production by proteolytic systems. In Nef region 123–152,
Lucchiari-Hartz et al. (17) identified naturally processed
peptides and the generation by 20S proteasomes from T1 cells of five
epitopes from the fragment Nef123–152 (128–135,
128–137, 135–143, 136–145, and 136–146), presented by HLA-A2 and
HLA-B7 molecules. We tested epitopes 135–143 and 128–137, which were
frequently recognized by T cells of our HIV-1-infected patients. All
these data suggest that ranking of immunodominance of T cell responses
to these peptides may depend on proteasome cleavage.
By using minigenes that code for 17–22-mer peptides and include one or several epitopes, different authors have shown the effect of COOH-terminal flanking residues on epitope liberation (3). According to data of Shimbara et al. (20), cleavage patterns of large peptides obtained with isolated proteasomes revealed that flanking Ala, basic, or hydrophobic residues enhance digestion, whereas flanking Gly, Pro, or acidic residues may inhibit it or prevent random cleavage and, thus, contribute to the efficient production of HLA-B7 ligands. This rule can be applied to Pro-rich Nef regions 68–85 and 128–143 where a large number (nine) of HLA-B7/B35-specific epitopes was detected. The abundance of hydrophobic and aromatic residues in central Nef region increases the chances of cleavage. Degradation of the 123–152 region by proteasome produces several fragments that are epitopes (17), and degradation of the 66–100 region probably generates numerous epitopes or their precursors.
It was then possible to define the relationships that may exist between
Nef structure and epitope liberation. The structure of HIV-1 Nef in
solution, described by Grzesiek et al. (11), comprises one
polyproline helix (residues 69–78), three other helices
(residues 81–94, 105–118, and 194–198), five -sheet strands
(residues 100–102, 126–128, 134–137, 142–146, and 181–186), and
two solvent-exposed long disordered loops (NH2
terminus 1–67 and 146–179). An abundance of hydrophobic, aromatic,
and basic residues is compatible with high concentration of HLA-binding
motifs, structural formations such as -sheets or helices, and
efficient proteasome digestion. In fact, regions 66–100 and 126–146
are both rich in HLA-binding motifs and well-structured segments of
helices (69–78 and 81–94), or -sheets (126–128, 134–137,
142–146), and their degradation by proteasomes delivers epitopes (this
study and Ref. 17). In contrast, from the Los Alamos
database (21), no CTL epitopes were identified in the
coiled loop region 20–67, which is very rich in small residues (Ala,
Thr, and Ser) and in charged residues (Arg, Lys, Asp, and Glu), or in
region 146–179, which is very rich in negatively charged (Asp and Glu)
residues. These two regions are poor in HLA-binding residues and are
thus unlikely to be a source of CTL epitopes.
It remains to be confirmed whether the potential of any protein to generate CTL epitopes can be predicted more precisely by analyzing both secondary and tertiary structures.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeannine Choppin, Institut National de la Santé et de la Recherche Médicale, Unité 445, Institut Cochin de Génétique Moléculaire, Hôpital Cochin, 27 rue du faubourg Saint-Jacques, 75014 Paris, France.
3 Abbreviations used in this paper: 2m, 2-microglobulin; AU, absorbance unit; ELISPOT, enzyme-linked immunospot; SFC, spot-forming cells.
Received for publication July 5, 2000. Accepted for publication March 8, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% binding at 10-6 M) are represented with a bold
line, peptides with moderate reactivity (20 to <50% at
10-6 M) with a normal line, and peptides with low
reactivity (<20% at 10-6 M and significant binding at
10-4 M) with a dotted line. Binder-rich regions 66–100
and 126–146 are delimited by arrows. *, Peptide 136–146 was
described as an HLA-A2 binder (
