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Division of
*
Viral Pathogenesis, Harvard Medical School, Beth Israel-Deaconess Medical Center, Boston, MA 02215; and
Antibody Department, Beckman-Coulter Immunotech, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SIVmac-infected rhesus monkeys provide a valuable system in which to explore CTL immunity to AIDS viruses. Monkeys expressing the MHC class I allele Mamu-A*01 (1) develop a reproducible dominant Gag-specific CTL response following infection with SIVmac. This Mamu-A*01-restricted CTL response is directed against a single 9-aa Gag epitope, CTPYDINQM, known as p11C(C-M) (2). Exploring the ramifications of variability at that epitope for CTL recognition, Shen et al. (3) observed that synthetic peptides containing single amino acid substitutions at positions 2, 5, 7, 8, and 9 were similar in their ability to sensitize B-lymphoblastoid cell line (B-LCL)3 targets for CTL lysis. In contrast, substitution of the amino acids at positions 4 or 6 resulted in a significant decrease in target sensitization, and peptides substituted at position 3, the anchor residue, were ineffective at sensitizing targets. These data suggested that considerable variability in this epitope sequence is tolerated without loss of CTL recognition.
Studies were initiated to investigate further the capacity of CTL to respond to variations in the p11C(C-M) sequence. In pursuing this work, we have found that subtle differences in the fine specificity of the CTL response allow cross-recognition of peptide variants that have not previously been presented to the immune system. This finding suggests that epitope selection evolves to permit recognition of a considerable degree of viral sequence variation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rhesus monkeys used in this study were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee for Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (4). Monkeys were screened for the presence of the Mamu-A*01 allele using a PCR-based technique as previously described (5, 6). DNA sequence analysis was performed on all potential positive samples to confirm identity with the established prototype Mamu-A*01 sequence (1).
DNA immunizations and viruses
Some of the monkeys used in the current study were part of a larger study of CTL responses to plasmid DNA vaccination with or without cytokine augmentation. Details of the DNA vaccination methods have been published elsewhere (7, 8). Briefly, monkeys were immunized i.m. in the quadriceps and/or deltoids with 4–5 mg per injection site of CsCl-purified plasmid DNA using a needleless, CO2-powered Biojector 2000 delivery device (Bioject, Portland, OR). Each injection consisted of either the single plasmid, pV1R-SIVgag (7, 8, 9), encoding a codon-optimized SIVmac239 gag, or a combination of plasmids including pV1R-SIVgag and pV1J-HIVenv (encoding a codon-optimized HIV1 89.6) (10, 11) with or without a plasmid encoding Ig-linked IL-2 (12, 13). Monkeys used in the plasmid ± cytokine study (7) were subsequently challenged with the SIV/HIV chimeric virus SHIV89.6P. Data shown in the current study as examples of "DNA-vaccinated monkeys" are from studies done before viral challenge of these monkeys. Data used in the current study as examples of "chronically SIV-infected monkeys" are from rhesus monkeys infected with SIVmac251 for greater than 1 year before study.
Abs, tetramers, and peptides
PE-conjugated mouse anti-human TCR V
23 Ab (clone AF23)
was obtained from Beckman-Coulter Immunotech (Marseille, France)
and used at a concentration of 0.5 µg/100 µl sample.
FITC-conjugated mouse anti-human V3.1 Ab (clone 8F10) was
obtained from Endogen (Woburn, MA) and used at a concentration of 1
µg/100 µl sample. The following mAbs were also used: PE-Texas
Red-conjugated anti-CD8
(clone 7PT3F9; Beckman Coulter),
PE-Texas Red-conjugated anti-CD8 (Beckman Coulter),
FITC-conjugated anti-CD3 (clone FN18; BioSource International,
Camarillo, CA), or FITC-conjugated anti-CD3 (clone SP34; BD
PharMingen, San Diego, CA). Tetrameric complexes of Mamu-A*01 bound to
peptides p11C(C-M), mp4, mp6, or p54AS were prepared as described
previously (14) using fluorescently labeled streptavidin
conjugates of PE or allophycoerythrin from Prozyme (San Leandro, CA) or
PerCP (a gift of Holden Maecker, BD Biosciences, Mountain View, CA).
Peptides, synthesized as nonamers and purified to 90% homogeneity,
were obtained from QCB/Biosource (Hopkinton, MA). Lyophilized peptides
were dissolved in a minimum volume of DMSO (Sigma-Aldrich, St. Louis,
MO) and diluted to a stock peptide concentration of 15 mg/ml in water
containing 5 mM DTT (Sigma-Aldrich), then frozen at -70°C in
aliquots. Before use, peptides were diluted to working concentration in
RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with glutamine,
12% FCS, penicillin, streptomycin, and gentamicin.
Cytotoxicity assays
PBMC from Mamu-A*01+ rhesus
monkeys were seeded on day 0 in 12-well plates at a density of 7
million cells/well and grown for 3 days in RPMI 1640 medium
supplemented with glutamine, 25 mM HEPES (Calbiochem, San Diego, CA),
12% FCS, penicillin, streptomycin, gentamicin, and the appropriate
stimulating peptide. We have found that the concentration of p11C(C-M)
peptide which results in half-maximal stimulation and target
sensitization is 30–50 ng/ml (data not shown). The mp4 and mp6
peptides both bind Mamu-A*01 with 3.5- to 5-fold lower
affinity than p11C(C-M) and require an 
1 log higher concentration to
sensitize targets (data not shown). However, both are comparable to
p11C(C-M) in the concentration required for half-maximal stimulation
(data not shown). For the present studies, p11C(C-M) was used at a
concentration of 100 ng/ml, and mp4 and mp6 were used at 20 µg/ml.
Recombinant human IL-2 (Hoffmann-LaRoche, Nutley, NJ) was added on day
3 to a final concentration of 10 U/ml. After 10–14 days of culture,
effector cells were assessed for cytotoxic activity against
51Cr-labeled, peptide-sensitized autologous B-LCL
targets. Plates were incubated for 4 h in a humidified
CO2 incubator at 37°C. Specific chromium
release was calculated as (experimental release - spontaneous
release) / (maximum release - spontaneous release). Percentage of
specific lysis was calculated as (specific release for test
peptide - specific release for control peptide, p11B
(ALSEGCTPYDIN)) x 100. All wells were assayed in duplicate or
triplicate.
Flow cytometry
PBMC cultured in the presence of peptide and IL-2 were harvested on days 10–14 and separated over a Ficoll layer (Ficoll-Paque Plus; Amersham-Pharmacia Biotech, Uppsala, Sweden). Cultured cells or whole-blood specimens were stained with mp4, mp6, or mock tetramer for 15 min at room temperature followed by addition of the second tetramer (p11C(C-M) or mock) for 15 min at room temperature. Cells were then stained with a mixture of anti-CD3 and anti-CD8 Abs for 15 min. Cultured cells were fixed in 1% formaldehyde. Whole-blood specimens were lysed using a Coulter Immunoprep reagent system and a Q-prep workstation (Beckman-Coulter) before fixing in 1.5% formaldehyde. Samples were analyzed on a Coulter EPICS XL-MCL or a FACSCaliber flow cytometer (BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC from Mamu-A*01+ rhesus monkeys
chronically infected with SIVmac were assessed for functional
cytotoxicity directed against the dominant Mamu-A*01-restricted Gag
epitope p11C(C-M) (CTPYDINQM), or two peptide variants, each differing
from p11C(C-M) by a single amino acid. As shown for one monkey in Fig. 1
A, PBMC stimulated with
p11C(C-M) effectively lysed autologous target cells pulsed with
p11C(C-M) or either of the two variant peptides, mp4 (CTPIDINQM) or mp6
(CTPYDYNQM). In contrast, mp4-stimulated effectors mediated minimal
cytotoxicity when tested against mp6-pulsed targets, although these
effectors readily lysed mp4- or p11C(C-M)-sensitized targets.
Similarly, mp6-stimulated effectors mediated minimal cytotoxicity when
tested against mp4-pulsed targets, but readily lysed mp6- and
p11C(C-M)-sensitized targets.
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, B and C). Effectors stimulated with p11C(C-M)
preferentially lysed mp6- or p11C(C-M)-sensitized targets and exhibited
only weak to moderate cytotoxic activity for mp4-sensitized targets.
However, in both of these cases, stimulation of PBMC with mp4 expanded
effector populations that mediated moderate lysis of mp4- and
p11C(C-M)-sensitized targets and minimal lysis of mp6-sensitized
targets. Similarly, mp6 stimulation of PBMC expanded effector
populations that mediated lysis of mp6- and p11C(C-M)-sensitized
targets but not mp4-sensitized targets. These data support the notion
that the dominant p11C(C-M)-directed CTL response of monkeys infected
with SIV can be functionally subdivided into components with distinct
fine specificities. MHC class I tetramers can be used to distinguish subpopulations of epitope-specific CD8+ T cells with differing fine specificities
To determine whether MHC class I/peptide tetramers might
discriminate CD8+ T cells with specificities for
closely related epitopes, peptide/Mamu-A*01 tetramers were constructed
using the nonamer p11C(C-M), mp4, and mp6 peptides. PBMC from
SIVmac-infected Mamu-A*01+ rhesus monkeys
were stimulated with p11C(C-M) and stained with the p11C(C-M)-, mp4-,
or mp6-tetramers. As a control, a tetramer was used that was
constructed with Mamu-A*01 and the unrelated Mamu-A*01-restricted
peptide p54AS (TVPWPNASL) (15, 16). As shown in Fig. 2A, p11C(C-M)-stimulated cells
(Fig. 2A, filled bars) were readily detected with the mp4
tetramer (Fig. 2A, left) and with the p11C(C-M)
tetramer (Fig. 2A, middle). Neither tetramer
detected p54AS-stimulated cells, but p54AS-stimulated cells were
detected by the control p54AS tetramer (Fig. 2A, hatched
bar, right). Thus, both the p11C(C-M) and the mp4 tetramers
could discriminate CD8+ T cells with specificity
for an unrelated Mamu-A*01-restricted peptide. As shown in Fig. 2B, the mp6 and p11C(C-M) tetramers could also discriminate
cells from a second monkey with specificity for the unrelated
Mamu-A*01-restricted epitope.
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, cells stimulated with mp4 stained
exclusively with the mp4 tetramer, while cells stimulated with mp6
stained exclusively with the mp6 tetramer. In contrast,
p11C(C-M)-stimulated cells stained with both tetramers. Importantly,
cells stimulated in vitro with mp4, mp6, or p11C(C-M) all stained with
the p11C(C-M) tetramer (data not shown). These data are in agreement
with the functional CTL data showing that CD8+ T
cells can discriminate between closely related peptides presented by
the Mamu-A*01 molecule (Fig. 1A).
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A, filled bars) varied
between 0.22 and 9.9%. Interestingly, in monkeys 138 and 191, and to a
lesser extent in monkey 94, unstimulated lymphocytes stained strongly
with the mp6 tetramer (Fig. 4A, hatched bars) but only
weakly with the mp4 tetramer (Fig. 4A, open bars). In
unstimulated lymphocytes of monkeys 403 and 348, the opposite pattern
was observed (Fig. 4A). Because the total number of
tetramer-staining cells in fresh whole blood is generally low, PBMC
from the same monkeys were similarly assessed after these cells were
expanded by stimulation in vitro with p11C(C-M). As shown in Fig. 4B, the fraction of the cultured
CD3+CD8+ lymphocytes that
stained with the p11C(C-M) tetramer (Fig. 4B, filled bars)
varied between 15 and 82%. As in the studies of the freshly obtained
whole blood, a larger proportion of the cultured lymphocytes from
monkeys 138, 191, and 94, as well as from the additional monkey 253,
stained with the mp6 tetramer (Fig. 4B, hatched bars) than
with the mp4 tetramer (Fig. 4B, open bars), while in
cultured cells from monkeys 403 and 348, mp4 tetramer-binding cells
outnumbered the mp6 tetramer-binding cells. Thus, consistent with the
functional CTL studies (Fig. 1), these tetramer studies indicate that
the p11C(C-M)-specific CD8+ T cell response of
the Mamu-A*01+ SIVmac-infected monkeys
appears to be biased toward mp4 in some animals and toward mp6 in
others.
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One possible explanation for the preferential staining of
CD8+ T lymphocytes in fresh blood by the mp6 or
mp4 tetramers and the preferential expansion of mp6- or mp4-specific
CTL after in vitro stimulation with p11C(C-M) is that SIVmac variants
expressing mutations in the p11C(C-M) epitope evolve in vivo, and that
these viral variants, in turn, cause preferential expansion of selected
CTL subpopulations. If this hypothesis were correct,
Mamu-A*01+ rhesus monkeys immunized with a
nonreplicating wild-type SIVmac Gag immunogen would not be expected to
exhibit a bias in mp4 or mp6 recognition. We examined the p11C(C-M)-,
mp4-, and mp6-specific responses of seven
Mamu-A*01+ rhesus monkeys that were immunized
with a plasmid DNA vaccine expressing a codon-optimized SIVmac239
gag gene. These animals were being used in a study assessing
the immunity to SIV induced by cytokine-augmented plasmid DNA
vaccination (13). The p11C(C-M)-specific
CD8+ T cell responses of these animals, as
measured by fresh-blood tetramer staining, were too low (0.0–1.2% of
CD3+CD8+ cells, Fig. 5A) to allow reliable
quantitation of the unstimulated mp4 and mp6 tetramer-binding cell
populations (data not shown). However, after in vitro stimulation with
p11C(C-M), 16–71% of the CD8+ peripheral blood
T cells from these animals could be stained with p11C(C-M) tetramer. As
shown in Fig. 5A, the fraction of p11C(C-M) tetramer-binding
CD8+ T cells detected after in vitro expansion
correlated closely with the fraction of tetramer-binding
CD8+ T cells detected in fresh blood. When these
p11C(C-M)-stimulated lymphocytes were stained with the mp4 and mp6
tetramers, biases in the mp4 and mp6 tetramer binding were detected. As
shown in Fig. 5B, a substantial percentage of the
p11C(C-M)-stimulated PBMC from monkeys 483, 728, 811, 820, and 833
stained with the mp6 tetramer but not with the mp4 tetramer.
CD8+ T cells from monkey 893 cells stained with
the mp4 but not the mp6 tetramer. In the CD8+ T
cells from monkey 702, staining with the mp4 and mp6 tetramers was
approximately equal. Thus, the CD8+ T cell
response elicited by the p11C(C-M) sequence is usually biased toward
either mp4 or mp6, and the establishment of this bias does not depend
upon the evolution of the epitope sequence.
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Based on the above observations, we hypothesized that the mp4- and
mp6-specific CD8+ T cells detected in tetramer
assays and in functional CTL assays were mono- or oligoclonal subsets
of the polyclonal population of p11C(C-M)-specific
CD8+ T cells. To test this hypothesis, we sought
to perform simultaneous tetramer staining with the p11C(C-M) tetramer
and either the mp4 or mp6 tetramer. All of these tetramers were
predicted to bind to the same target, the TCR, with perhaps different
affinities and avidities. Therefore, it was necessary first to
establish empirically the subsaturating concentrations of each tetramer
for use in staining these lymphocytes to maximize the mean fluorescence
intensity per cell of the tetramer-binding lymphocytes, while at the
same time avoiding the use of concentrations of a tetramer that were so
high as to inhibit competitively the binding of the other tetramer. The
concentration of p11C(C-M) tetramer used for staining in this study
resulted in a 
0.3-log decrease in mutant peptide tetramer mean
fluorescence intensity compared with cells stained with mutant peptide
tetramer alone. Similarly, the concentration of mutant peptide
tetramers used for staining in this study resulted in a 0.15-log
decrease in p11C(C-M) tetramer mean fluorescence intensity compared
with cells stained with p11C(C-M) tetramer alone (data not shown). It
is likely that, at the concentrations of tetramers used, only a
fraction of the available binding sites were occupied. However,
staining with higher concentrations of tetramers did result in
significant competition for binding (data not shown). These results
support the notion that all three tetramers were binding to a common
receptor site.
A representative example of double tetramer staining of
p11C(C-M)-stimulated cultured PBMC from an SIVmac-infected
Mamu-A*01+ monkey is shown in Fig. 6. As predicted, the p11C(C-M)
tetramer-binding CD8+ T lymphocytes could be
subdivided into mp4 tetramer-positive and mp4 tetramer-negative subsets
(Fig. 6A), as well as into mp6 tetramer-positive and mp6
tetramer-negative subsets (Fig. 6B). Importantly, we did not
identify any mp4-specific or mp6-specific CD8+ T
cells that did not also stain with the p11C(C-M) tetramer.
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). Similarly, in the PBMC of the three
animals previously identified as having a high frequency mp4-specific
CD8+ T cell response, the majority of p11C(C-M)
tetramer-binding CD8+ T lymphocytes also bound
mp4 tetramer. Taken together, these data show that the
CD8+ T cell response to p11C(C-M) in both
SIVmac-infected and plasmid DNA-immunized
Mamu-A*01+ rhesus monkeys is functionally
polyclonal, and subtle differences in the fine specificity of the
p11C(C-M)-specific CD8+ T cells can be detected
by both functional and tetramer analyses.
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variable regions than those
p11C(C-M)-specific CD8+ T lymphocytes that recognize mp6
We reasoned that these differences in fine specificity may result
from differences in the TCRs expressed by the different
p11C(C-M)-specific lymphocyte subpopulations. To demonstrate
differences in the TCRs of these cells, we used a panel of human
TCR-specific mAbs that cross-react with epitopes on rhesus monkey T
cells (W. A. Charini, J. E. Schmitz, and A. Necker,
unpublished observations; data available on-line at
http://research.bidmc.harvard.edu/V_path/v_pathogens.asp) for these
studies. Fresh peripheral blood was screened for the presence of
CD8+ T cells that stained with both the p11C(C-M)
tetramer and one of several V-specific Abs. PBMC from all 13 monkeys
described in this study bound the p11C(C-M) tetramer and each of the
V Abs tested. In two monkeys, a small subset of PBMC were observed
to stain with both the p11C(C-M) tetramer and one of the V-specific
mAbs, simultaneously. PBMCs from these two monkeys were then stimulated
in vitro with mp4 or mp6 peptides and then stained with the
corresponding V Ab together with either the mp4 or mp6 tetramers. As
shown in Table I, 78% of the mp6
tetramer-staining PBMC of monkey 253 stained with anti-V23,
whereas only 8.1% of the mp4 tetramer-staining cells stained with the
same mAb. This finding indicates a preference for V23 usage among
CD8+ T cells specific for mp6 in this animal. In
PBMC of monkey 712, a significant proportion (14%) of the mp6
tetramer-binding, but less than 1% of the mp4 tetramer-binding,
CD8+ T lymphocytes stained with the
anti-V3.1 mAb, indicating a preference for V3.1 usage among
some, but not all, CD8+ T cells specific for mp6
in this monkey. Moreover, these data indicate that the mp6
tetramer-specific T cells can be further subdivided into at least two
subfractions based on anti-V3.1 staining. With these reagents it
is thus possible not only to demonstrate the polyclonality of the
p11C(C-M)-specific CTL response, but also to begin to ascribe
functional differences in specificity to individual clonal
subpopulations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the three-dimensional structures of the TCR/peptide/MHC class
I complex have been solved by x-ray crystallography for a small number
of human and murine complexes (17, 18, 19), no data are yet
available for Mamu-A*01/p11C(C-M) or its TCR ligands.
Indirect evidence from functional assays using substituted peptides
(2, 3) and peptide elution and peptide binding assays
(2, 20) suggests that the proline at position three of the
p11C(C-M) sequence interacts with the Mamu-A*01 molecule and
serves as an anchor residue for the epitope peptide. This possibility
is consistent with the finding that all known
Mamu-A*01-binding peptides contain prolines at position
three (20). Other epitope peptide requirements for binding
to Mamu-A*01 inferred from a comparative analysis of
Mamu-A*01-binding peptides and direct testing of peptides
both in whole cell binding assays and intracellular IFN-
assays
include the preference for a small residue (T or S) at position two and
a hydrophobic or aromatic residue (I, L, V, M, or F) at the C terminus
(2, 20). Thus, side chains of the amino acids at positions
2 and 3, and the C terminus of the peptide are predicted to interact
with the Mamu-A*01 molecule, while the remaining amino acid
side chains, including those of the amino acids at positions 4 and 6,
are likely to interact with the TCR.
Understanding the clonal diversity of the CTL response is particularly important for vaccine design. A clonally diverse response to a viral epitope increases the likelihood that multiple divergent strains of AIDS viruses may be recognized by CTL elicited through immunization with only a single consensus epitope sequence. Moreover, the more diverse the clonal response of T cells in an infected individual, the fewer the possibilities for viral escape through variation of the epitope sequence.
The polyclonality of the CTL response to a single peptide epitope is not unexpected and has, in fact, previously been demonstrated for p11C(C-M) (21). Several studies have also demonstrated that individual T cell clones can recognize MHC complexed with any of a number of related peptides. Such TCR/peptide/MHC class I interactions can vary in their binding affinities and in the type of T cell signaling that results. Some peptides are strong activators of T cells, whereas others act as weak T cell agonists or even as T cell antagonists (22, 23). Given the promiscuity of TCR contacts with peptide/MHC class I complexes, it is not surprising that different clonal populations of T cells overlap in their recognition of particular MHC/peptide class I complexes.
It is well established that Ab responses are polyclonal. Indeed,
polyclonality is a critical prerequisite for affinity maturation.
Competition among B cells for Ag binding results in the positive
selection of those B cell clones with the highest affinity for Ag.
Recent studies suggest that an analogous form of competition may occur
among T cells to create a highly focused secondary CTL response
(24, 25, 26). Haanen et al. (26) have
demonstrated that when mice are sequentially infected with strains of
influenza differing by two amino acids in the region of a dominant CTL
epitope they develop CTL capable of recognizing both viral variants,
and these T cells express a limited subset of V gene segments.
However, infection of mice with both influenza variants,
simultaneously, did not elicit a population of dual-specific T cells,
but, instead, distinct populations of strain-specific T cells. These
results provide evidence that fine-tuning of the CTL response occurs
following secondary exposure to epitope variants by selective
stimulation of a subset of memory T cell clones. Indeed, Kedel et al.
(25) have provided direct evidence that competition among
T cells recognizing unrelated peptides restricted by the same MHC class
I molecule occurs at the level of access to the APC. Such a mechanism
may also underlie the selection of T cell clones recognizing pairs of
closely related peptides.
The results of the present study suggest that a dominant epitope-specific CTL response is heterogeneous enough to include clones that recognize that epitope as well as multiple variants of that epitope. The extent of the heterogeneity of the responses that have been characterized in detail using T cell clones or PBMC from immunized or virus-infected animals may, in fact, represent only a small fraction of the epitopes that can be recognized by these populations of effector cells. Using only two variant epitope peptides, we were able to discriminate four subtypes of CTL: those with specificity for p11C(C-M) and mp4 but not mp6; those with specificity for p11C(C-M) and mp6 but not mp4; those with specificity for mp4, mp6, and p11C(C-M) (in most animals, a small fraction of cells; data not shown); and those with specificity for p11C(C-M) but neither mp4 nor mp6 (data not shown). The relative representation of cells of each subtype varied from animal to animal and depended on the nature of the immunogen and the secondary stimulation used. If the present studies had been done with more than two variant peptides, we would likely have defined an even larger number of variant recognition patterns.
Indeed, each CTL clone can be defined functionally on the basis of its response to the immunizing and variant sequences. In responding strongly to some variants and weakly to others, each clone will have a unique profile. The ability of an individual’s immune system to recognize epitope variants is dependent on each CTL clone’s contribution to the total response. That clone’s contribution to the immune response is a function of both the fractional representation of the individual clone in the total epitope-specific response and the clone’s ability to recognize variant sequences. Thus, if an individual is immunized with a particular epitope sequence, a polyclonal CTL response emerges that includes clones with specificities for epitope variants that the individual’s immune system has not yet encountered.
When monkeys are immunized with an SIVmac gag DNA vaccine, they develop p11C(C-M)-specific CTL responses with the same heterogeneous fine specificity seen in the p11C(C-M)-specific CTL of chronically SIVmac-infected monkeys. Because the immunogen in these DNA-vaccinated monkeys is nonreplicating and, therefore, not subject to mutational selective pressure in the region encoding the p11C(C-M) epitope, we can be confident that the immunized monkeys have only been exposed to the single wild-type p11C(C-M) sequence, CTPYDINQM. Nevertheless, the vaccine-elicited CTL response includes cell clones that are cross-reactive with p11C(C-M) variants to which the monkeys have not been specifically primed. This observation suggests that cell clones exist in this population of CTL that are reactive with numerous other peptides differing from the wild-type sequence by one or multiple amino acids.
Following primary immunization, exposure of PBL to a peptide variant in
vitro results in an expansion of CTL reactive with that particular
amino acid sequence. Indeed, a focusing of the CTL population like that
described by Haanen et al. (26) can be seen in vitro when
PBMC from SIVmac-infected (Figs. 1 and 3, and Table I) or DNA-immunized
(data not shown) monkeys are cultivated in the presence of a variant
epitope peptide. In vitro stimulation with mp4 expands a population of
p11C(C-M)- and mp4/p11C(C-M)-specific CD8+ T
cells that exhibits minimal cross-reactivity with mp6. Similarly, with
mp6 stimulation, a population of p11C(C-M)- and mp6/p11C(C-M)-specific
CD8+ T cells is selectively expanded that
exhibits minimal cross-reactivity with mp4.
The present study supports the conclusions of Buseyne et al. (27) and Buseyne and Rivière (28) that recognition of several naturally occurring variants of sequences of an HIV CTL epitope can occur through a single TCR. Those authors also demonstrated that, in certain circumstances, promiscuous CTL recognition of exogenous peptides presented by allogeneic TCR can occur. These findings indicate a remarkable tolerance of the TCR for variations of its ligand, both the peptide component and the MHC component.
In conclusion, priming a CTL response to a dominant epitope simultaneously results in priming to variants of the dominant peptide sequence. Subsequent exposure to the original epitope sequence or one of its variants can result in a focusing of the CTL response with a relative increase in the representation of previously minor epitope-specific CTL populations. In this way, the immune response "anticipates" viral sequence variation. Such a mechanism places severe restrictions on the types of mutations that can lead to loss of CTL recognition.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William A. Charini, Division of Viral Pathogenesis RE-113, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address: wcharini{at}caregroup.harvard.edu
3 Abbreviation used in this paper: B-LCL, B-lymphoblastoid cell line.
Received for publication June 28, 2001. Accepted for publication September 4, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. Sen, W. A. Charini, R. A. Subbramanian, E. R. Manuel, M. J. Kuroda, P. A. Autissier, and N. L. Letvin Clonal Focusing of Epitope-Specific CD8+ T Lymphocytes in Rhesus Monkeys following Vaccination and Simian-Human Immunodeficiency Virus Challenge J. Virol., January 15, 2008; 82(2): 805 - 816. [Abstract] [Full Text] [PDF]
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L. E. Valentine, S. M. Piaskowski, E. G. Rakasz, N. L. Henry, N. A. Wilson, and D. I. Watkins Recognition of Escape Variants in ELISPOT Does Not Always Predict CD8+ T-Cell Recognition of Simian Immunodeficiency Virus-Infected Cells Expressing the Same Variant Sequences J. Virol., January 1, 2008; 82(1): 575 - 581. [Abstract] [Full Text] [PDF]
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C. Chung, W. Lee, J. T. Loffredo, B. Burwitz, T. C. Friedrich, J. P. Giraldo Vela, G. Napoe, E. G. Rakasz, N. A. Wilson, D. B. Allison, et al. Not All Cytokine-Producing CD8+ T Cells Suppress Simian Immunodeficiency Virus Replication J. Virol., February 1, 2007; 81(3): 1517 - 1523. [Abstract] [Full Text] [PDF]
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E. R. Manuel, W. A. Charini, P. Sen, F. W. Peyerl, M. J. Kuroda, J. E. Schmitz, P. Autissier, D. A. Sheeter, B. E. Torbett, and N. L. Letvin Contribution of T-Cell Receptor Repertoire Breadth to the Dominance of Epitope-Specific CD8+ T-Lymphocyte Responses J. Virol., December 15, 2006; 80(24): 12032 - 12040. [Abstract] [Full Text] [PDF]
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M. S. Seaman, L. Xu, K. Beaudry, K. L. Martin, M. H. Beddall, A. Miura, A. Sambor, B. K. Chakrabarti, Y. Huang, R. Bailer, et al. Multiclade Human Immunodeficiency Virus Type 1 Envelope Immunogens Elicit Broad Cellular and Humoral Immunity in Rhesus Monkeys J. Virol., March 1, 2005; 79(5): 2956 - 2963. [Abstract] [Full Text] [PDF]
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 D. M. McKinney, R. Skvoretz, B. D. Livingston, C. C. Wilson, M. Anders, R. W. Chesnut, A. Sette, M. Essex, V. Novitsky, and M. J. Newman Recognition of Variant HIV-1 Epitopes from Diverse Viral Subtypes by Vaccine-Induced CTL J. Immunol., August 1, 2004; 173(3): 1941 - 1950. [Abstract] [Full Text] [PDF]
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A. R. Lopes, A. Jaye, L. Dorrell, S. Sabally, A. Alabi, N. A. Jones, D. R. Flower, A. De Groot, P. Newton, R. M. Lascar, et al. Greater CD8+ TCR Heterogeneity and Functional Flexibility in HIV-2 Compared to HIV-1 Infection J. Immunol., July 1, 2003; 171(1): 307 - 316. [Abstract] [Full Text] [PDF]
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F. Villinger, A. E. Mayne, P. Bostik, K. Mori, P. E. Jensen, R. Ahmed, and A. A. Ansari Evidence for Antibody-Mediated Enhancement of Simian Immunodeficiency Virus (SIV) Gag Antigen Processing and Cross Presentation in SIV-Infected Rhesus Macaques J. Virol., December 6, 2002; 77(1): 10 - 24. [Abstract] [Full Text] [PDF]
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A. Castilleja, D. Carter, C. L. Efferson, N. E. Ward, K. Kawano, B. Fisk, A. P. Kudelka, D. M. Gershenson, J. L. Murray, C. A. O'Brian, et al. Induction of Tumor-Reactive CTL by C-Side Chain Variants of the CTL Epitope HER-2/neu Protooncogene (369-377) Selected by Molecular Modeling of the Peptide: HLA-A2 Complex J. Immunol., October 1, 2002; 169(7): 3545 - 3554. [Abstract] [Full Text] [PDF]
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A. Boissonnas, O. Bonduelle, A. Antzack, Y.-C. Lone, C. Gache, P. Debre, B. Autran, and B. Combadiere In Vivo Priming Of HIV-Specific CTLs Determines Selective Cross-Reactive Immune Responses Against Poorly Immunogenic HIV-Natural Variants J. Immunol., October 1, 2002; 169(7): 3694 - 3699. [Abstract] [Full Text] [PDF]
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), mp6
(
), or p11C(C-M) (
). Data shown are specific lysis of
51Cr-labeled targets at a 16:1 E:T ratio.
