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J Virol. 2009 October; 83(19): 9803–9812.
Published online 2009 July 29. doi:  10.1128/JVI.00776-09
PMCID: PMC2748000

Diverse Cross-Reactive Potential and Vβ Gene Usage of an Epitope-Specific Cytotoxic T-Lymphocyte Population in Monkeys Immunized with Diverse Human Immunodeficiency Virus Type 1 Env Immunogens[down-pointing small open triangle]

Abstract

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vβ repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8+ T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8+ T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vβ gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.

The ability of the immune system to contain the replication of human immunodeficiency virus type 1 (HIV-1) is complicated by the extraordinary capacity of the virus to generate mutations (19). Mutations are rapidly generated by the replicating virus because of its inefficient reverse transcriptase (27) and its tendency to recombine with related virus forms (34). Certain newly generated viruses are selected because of a replication fitness advantage (4, 7, 10). Importantly, the accrual of mutations by replicating HIV-1 also provides a survival advantage to the virus in that it facilitates virus escape from immune recognition (4, 12, 21, 25). Much of the sequence heterogeneity in circulating strains of HIV-1 has arisen because of this ongoing genetic evolution of the virus (6).

The definition of how CD8+ T lymphocytes recognize mutant and divergent forms of HIV-1 will inform both our understanding of the immunopathogenesis of HIV-1 infections and strategies for developing an effective HIV-1 vaccine (22). CD8+ T lymphocytes play a central role in containing HIV-1 replication during the period of primary infection (5, 15, 17, 23) and in chronically infected individuals (9, 11, 24, 30). The diversity of variant viruses recognized by individual clones of CD8+ T lymphocytes should impact the immune control of HIV-1. Moreover, the clonal diversity of an HIV-1-specific CD8+ T-lymphocyte response either generated by vaccination or elicited by the replicating virus should contribute to containing the emergence of mutant forms of the virus that can evade immune recognition (22, 26, 31).

The present studies were initiated to characterize at a clonal level CD8+ T-lymphocyte recognition of variant forms of the HIV-1 envelope (Env) in vaccinated and infected rhesus monkeys. These studies were done with monkeys selected to express the major histocompatibility complex (MHC) class I allele Mamu-A*01, and the Env region studied was the well-characterized HIV-1 V3 loop epitope p41A (8). CD8+ T-lymphocyte recognition of variant forms of the p41A epitope was evaluated using tetramers constructed with Mamu-A*01 and variant epitope peptides, and the clonal constituents of the responding CD8+ T lymphocytes were assessed by T-cell receptor Vβ (TCR Vβ) repertoire analysis and selected complementarity-determining region 3 (CDR3) sequencing. These studies demonstrate that monkeys sharing the same MHC class I molecule and receiving the same vaccine immunogens can elicit p41A epitope peptide-specific cytotoxic T-lymphocyte populations which exhibit differences in the ability to cross-react to variant epitopes.

MATERIALS AND METHODS

Immunizations and challenge of rhesus monkeys.

Seven adult Indian-origin rhesus monkeys (Macaca mulatta) were maintained in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care 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 (23a). All monkeys expressed the MHC class I allele Mamu-A*01, as determined by PCR-based typing using allele-specific primers, as previously described (14, 17).

Monkeys were immunized using a DNA prime/recombinant adenovirus (rAd) boost vaccine regimen, including both SIVmac239 Gag-Pol-Nef and HIV-1 Env immunogens. A group of three monkeys (AW13, AW2P, and AW28) was vaccinated with 89.6P Env as previously described (29). Two monkeys were vaccinated with HxB2 Env (419 and VFA), and two others were vaccinated with clade C Env (414 and KPA), as previously described (31).

These monkeys were challenged intravenously with 1 × 108 RNA copies of either simian-human immunodeficiency virus (SHIV)-89.6P (n = 4), as previously described (31), or the KB9 clone of SHIV-89.6P (n = 3).

Antibodies, tetramers, and peptides.

Antibodies used in this study were directly coupled to fluorescein isothiocyanate or PerCP Cy5.5. The following monoclonal antibodies were used: fluorescein isothiocyanate-conjugated anti-CD8 αβ (clone SK1; Becton Dickinson Pharmingen) and PerCP Cy5.5-conjugated anti-CD3 (clone SP34.2, Becton Dickinson Pharmingen). Mamu-A*01/HxB2-p41A/β2m, Mamu-A*01/clade C-p41A/β2m, and Mamu-A*01/89.6P-p41A/β2m tetramer complexes were prepared as previously described (8, 16). Streptavidin R-phycoerythrin or streptavidin-allophycocyanin (PhycoLink; ProZyme) were mixed stepwise with biotinylated Mamu-A*01/epitope-peptide/β2m complexes at a molar ratio of 1:4 to produce the tetrameric complexes.

HxB2-p41A ([YAPPISGQI]), clade C-p41A ([YAPPIAGNI]), and 89.6P-p41A ([YAPPITGQI]) were obtained from New England Peptide, LLC. Lyophilized peptides were dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO), diluted to a stock peptide concentration of 10 mg/ml in water, and then frozen at −80°C in aliquots. Before use, peptides were diluted to a working concentration in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with glutamine, 12% fetal calf serum, streptomycin, and gentamicin.

IFN-γ ELISPOT assays.

Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays were performed as previously described (18). Fresh peripheral blood mononuclear cells (PBMC) were plated in triplicate at 2 × 105 cells/well in a 100-μl final volume with either medium alone or the noted p41A variant peptides at a concentration of 1 μg/ml. The mean number of spots from triplicate wells was calculated for each animal and adjusted to represent the mean number of spots per 106 PBMC.

Flow cytometry.

PBMC were isolated from monkeys 27 to 290 weeks following DNA prime/rAd boost immunization and 20 to 28 weeks following SHIV-89.6P challenge, separated over a Ficoll layer (Ficoll-Paque Plus; Amersham Pharmacia Biotech), and cryopreserved.

Thawed PBMC cultured in the presence of interleukin-2 (IL-2) were stimulated by HxB2-p41A, clade C-p41A, or 89.6P-p41A peptide (1 μg/ml), harvested on days 12 to 14, and separated using a preseparation filter (Miltenyi Biotec). The cells were stained with combinations of Mamu-A*01/HxB2-p41A/β2m, Mamu-A*01/clade C-p41A/β2m, and Mamu-A*01/89.6P-p41A/β2m tetramers conjugated with phycoerythrin or allophycocyanin for 15 min at room temperature and then stained with a mixture of anti-CD3 and anti-CD8 monoclonal antibodies for 15 min. Samples were analyzed on a FACSVantage SE flow cytometer (BD Biosciences). CD3+ CD8+ HxB2-p41A+, CD3+ CD8+ clade C-p41A+, or CD3+ CD8+ 89.6P-p41A+ epitope-specific T-lymphocyte populations were sorted to a purity of at least 98%.

Generation of cDNA.

RNA was extracted from either CD3+ CD8+ HxB2-p41A+, CD3+ CD8+ clade C-p41A+, or CD3+ CD8+ 89.6P-p41A+ epitope-specific T-lymphocyte populations, according to the instructions supplied with the RNeasy extraction minikit from Qiagen. cDNA was then synthesized from the extracted RNA, as outlined in the Super SMART PCR cDNA synthesis kit from Clontech Laboratories. Briefly, the single-stranded cDNA reaction was catalyzed using Moloney murine leukemia virus reverse transcriptase with the 3′ SMART CDS primer II A and Smart II A oligonucleotide primers provided in the Super SMART cDNA synthesis kit. Preamplified, double-stranded cDNA libraries were made using 10 to 25 cycles of PCR amplification, utilizing the 5′ PCR primer II A and reagents also provided in the Clontech kit. The optimal number of cycles of preamplification was determined by performing a test run in the presence of SYBR green to determine the maximum number of PCR cycles that could be performed in the log-linear amplification (20).

Primers and sequencing.

High-performance liquid chromatography-purified primers used for the real-time PCR assay and sequencing (32) were obtained from BioSource International. Real-time TaqMan probes were synthesized at Biosearch Technologies, Inc. Primers specific for the variable and constant region of the TCRβ chain were designed from rhesus monkey TCR sequences obtained from GenBank and generated in our laboratory.

Quantitative PCR.

cDNA derived from each sample was equally distributed into 48 individual PCRs. Each reaction contained a sense Vβ family-specific primer, an antisense Cβ-specific primer, and the TaqMan Cβ probe. PCRs were carried out using SureStart Taq (Stratagene). The real-time PCR was carried out for 50 cycles on an Mx4000 quantitative PCR machine (Stratagene) under the following conditions: 95°C for 10 min and 50 cycles of 95°C for 10 s, 58°C for 30 s, read fluorescence, and 72°C for 30 s. Background values for these Vβ quantitative PCR assays were, on average, 0.7% of the total copies in a series of reactions. Thus, we chose 0.7% as the threshold for considering a particular Vβ gene family as contributing to the effective Vβ repertoire of an epitope-specific CD8+ T-cell response.

Sequencing.

The CDR3 of Vβ 7B and Vβ 13C.2 in cDNA samples from CD3+ CD8+ clade C-p41A+- and from CD3+ CD8+ 89.6P-p41A+-sorted T cells were sequenced. cDNA generated for use in the quantitative PCR assays was used as a template for a second round of PCRs utilizing Vβ 7B or Vβ 13C.2 primers and a Cβ primer (BioSource International, Inc.). The cDNA was amplified for 35 cycles in a Perkin-Elmer 9600 GeneAmp PCR system under the following conditions: 95°C for 10 min and 50 cycles of 95°C for 10 s, 58°C for 30 s, and 72°C for 30 s. PCR products were purified using the MinElute reaction cleanup kit (Qiagen) by following the instructions from the manufacturer, cloned using the pGEM-T Easy system (Promega), and sequenced using a Cβ antisense primer for the determination of the CDR3 sequence. The CDR3 lengths were expressed as numbers of amino acids spanning the VDJ-joining segments.

RESULTS

Breadth of reactivity of Env-specific CD8+ T lymphocytes from HIV-1 89.6P Env-vaccinated and SHIV-89.6P-infected Mamu-A*01+ rhesus monkeys.

In view of the importance of the breadth of T-lymphocyte reactivity to contain the replication of variant forms of HIV-1, we sought first to characterize the cross-reactivity of CD8+ T lymphocytes of both 89.6P Env-vaccinated and SHIV-89.6P-infected Mamu-A*01-positive (Mamu-A*01+) rhesus monkeys. To facilitate these comparisons, we evaluated the CD8+ T-lymphocyte responses to three variant forms of the Mamu-A*01-restricted HIV-1 Env epitope p41A, consisting of two clade B epitope peptides (89.6P-p41A and HxB2-p41A) and one clade C epitope peptide (clade C-p41A) (Table (Table1).1). PBMC from three 89.6P Env-vaccinated monkeys (AW13, AW28, and AW2P) were stimulated in vitro with either HxB2-p41A, 89.6P-p41A, or clade C-p41A epitope peptide, and each of these in vitro-expanded lymphocyte populations was assessed for binding to tetramers constructed with these same variant epitope peptides (Fig. (Fig.1).1). Following the in vitro stimulation of PBMC with HxB2-p41A, which is a clade B epitope sequence that differed by one amino acid from that used to immunize these three monkeys, the expanded CD8+ T-lymphocyte populations bound tetramers constructed with both of the prototype clade B epitope peptides, the HxB2 and the 89.6P forms of p41A (Fig. (Fig.1A).1A). Following in vitro stimulation of the PBMC with the 89.6P-p41A epitope peptide, which is the same epitope peptide sequence used to immunize the monkeys, the expanded CD8+ T-lymphocyte populations bound tetramers constructed with the HxB2-form of p41A, and a small subset of these expanded CD8+ T lymphocytes bound a tetramer constructed with the more distantly related clade C-p41A peptide (Fig. (Fig.1B).1B). Finally, following in vitro stimulation of the PBMC with the clade C-p41A peptide, a small subset of the expanded CD8+ T-lymphocyte population bound to tetramers constructed with the 89.6P-p41A and HxB2-p41A peptides (Fig. (Fig.1C).1C). Therefore, immunization with one form of the p41A peptide expanded CD8+ lymphocytes that recognize diverse forms of this epitope peptide.

FIG. 1.
Cross-reactivity of p41A epitope-specific CD8+ T-lymphocyte populations in 89.6P Env-immunized Mamu-A*01+ rhesus monkeys. PBMC were isolated from monkeys AW13, AW28, and AW2P following vaccination. Cells were stimulated in vitro ...
TABLE 1.
Sequences of nonamer peptides used to stimulate and stain p41A epitope-specific CD8+ T lymphocytes

A similar study was performed on PBMC from the same three monkeys after they were challenged with the chimeric virus SHIV-89.6P (Fig. (Fig.2).2). Cross-reactivity of the p41A-specific CD8+ T-lymphocyte populations was observed that was comparable to that observed following vaccination of these monkeys. Most of the p41A-specific CD8+ T lymphocytes that recognized HxB2-p41A also recognized 89.6P-p41A, while only a small subset of the HxB2-p41A-specific or the 89.6P-p41A-specific CD8+ T lymphocytes recognized the clade C-p41A peptide. Therefore, the reactivity of CD8+ T cells from these monkeys did not broaden following infection.

FIG. 2.
Cross-reactivity of p41A epitope-specific CD8+ T-lymphocyte populations in 89.6P Env-immunized Mamu-A*01+ rhesus monkeys following SHIV-89.6P challenge. PBMC were isolated from monkeys AW13, AW28, and AW2P on day 142 following ...

To determine the particular TCR Vβ gene families that mediate the cross-recognition of the various forms of p41A, the p41A peptide-stimulated CD8+ T-lymphocyte populations were subjected to RNA extraction, cDNA synthesis, and Vβ analysis using rhesus monkey Vβ family-specific primer pairs (Fig. (Fig.3).3). In the setting of vaccination alone and following SHIV-89.6P challenge, p41A-specific CD8+ T lymphocytes from monkey AW13 that recognized 89.6P- and HxB2-p41A, but not clade C-p41A, made use of Vβ 12.3, 13A, 13C.1, 13C.2, 13C.3, and 15, while those CD8+ T lymphocytes that recognized all three forms of p41A made use of Vβ 04 and 23. CD8+ T lymphocytes from monkey AW28 that recognized 89.6P- and HxB2-p41A, but not clade C-p41A, made use of Vβ 13C.2, while those CD8+ T lymphocytes that recognized all three forms of p41A made use of Vβ 7B and 12.3. CD8+ T lymphocytes that recognized 89.6P- and HxB2-p41A, but not clade C-p41A, from monkey AW2P made use of Vβ 9A, 9C, 13C1, 13C2, and 15, while those CD8+ T lymphocytes that recognized all three forms of p41A made use of Vβ 13A. We were therefore able to define the TCR Vβ gene families for each monkey that mediated the cross-recognition of the various forms of p41A.

FIG. 3.
Vβ repertoire analysis of p41A epitope-specific CD8+ T-lymphocyte populations in 89.6P Env-immunized Mamu-A*01+ rhesus monkeys following vaccination and challenge. PBMC were isolated from monkeys AW13, AW28, and AW2P following ...

Breadth of reactivity of p41A-specific CD8+ T lymphocytes from HxB2 Env-vaccinated and SHIV-89.6P-infected Mamu-A*01+ rhesus monkeys.

We sought to expand our experience using tetramers and Vβ repertoire analysis to characterize the breadth of CD8+ T-lymphocyte recognition of variant epitope peptides. To this end, two Mamu-A*01+ rhesus monkeys (419 and VFA) were immunized with HxB2 Env, and peripheral blood CD8+ T lymphocytes from these monkeys were assessed for reactivity with tetramers constructed with Mamu-A*01 and the two clade B p41A Env peptides, HxB2- and 89.6P-p41A (Fig. (Fig.4,4, four left panels). As in the previously described study (Fig. (Fig.1),1), most of the vaccine-induced CD8+ T-lymphocyte population that recognized the HxB2-p41A peptide in association with Mamu-A*01 also recognized the 89.6P-p41A peptide in association with Mamu-A*01. These tetramer binding studies were consistent with functional T-lymphocyte responses elicited by the peptides. PBMC from both monkeys generated IFN-γ spot-forming cell (SFC) responses after in vitro exposure to HxB2-p41A, with a smaller magnitude of cross-reactive responses to clade C-p41A (Fig. (Fig.5A).5A). Following challenge with SHIV-89.6P, most of the CD8+ T lymphocytes that bound the HxB2-p41A tetramer also bound the 89.6P-p41A tetramer (Fig. (Fig.4,4, four right panels). Thus, most of the CD8+ T lymphocytes that recognized one of these clade B p41A peptides also recognized the other clade B p41A peptide.

FIG. 4.
Cross-reactivity of p41A epitope-specific CD8+ T-lymphocyte populations in HxB2 Env-immunized Mamu-A*01+ rhesus monkeys following vaccination and challenge. PBMC were isolated from monkeys 419 and VFA at week 27 following vaccination ...
FIG. 5.
p41A-specific IFN-γ ELISPOT responses of PBMC from Env-immunized Mamu-A*01+ rhesus monkeys. (A) Responses observed for HxB2 Env-immunized monkeys (VFA and 419). (B) Responses observed for clade C Env-immunized monkeys (414 and ...

To determine the particular TCR Vβ gene families that mediate the cross-recognition of HxB2-p41A and 89.6P-p41A, the p41A peptide-stimulated CD8+ T-lymphocyte populations were subjected to Vβ analysis (Fig. (Fig.6).6). Following vaccination alone and following virus challenge, p41A-specific CD8+ T lymphocytes from monkey 419 that recognized 89.6P-p41A and HxB2-p41A made use of the same Vβ genes: 2, 7B, 12.3, 13A, 13C.1, 13C.2, and 13C.3. Similarly, following vaccination alone and following virus challenge, p41A-specific CD8+ T lymphocytes from monkey VFA that recognized 89.6P-p41A and HxB2-p41A made use of the same Vβ genes: 2, 5B.1, 5B.1v, 6A, 12.3, 13A, 13C.1, 13C.2, 13C.3, 15, 16, and 21. Therefore, consistent with the cross-reactivity of these CD8+ T-lymphocyte populations, as determined by tetramer binding, the same Vβ genes were employed to mediate recognition of these peptides.

FIG. 6.
Vβ repertoire analysis of p41A epitope-specific CD8+ T-lymphocyte populations in HxB2 Env-immunized Mamu-A*01+ rhesus monkeys following vaccination and challenge. PBMC were isolated from monkeys 419 and VFA at week 27 following ...

Breadth of reactivity of p41A-specific CD8+ T lymphocytes from clade C Env-vaccinated and SHIV-89.6P-infected Mamu-A*01+ rhesus monkeys.

We then used this same technical approach to characterize the recognition of clade B p41A epitope sequences by CD8+ T cells primed with a divergent clade C Env immunogen. Two Mamu-A*01+ rhesus monkeys (KPA and 414) were immunized with clade C Env, and peripheral blood CD8+ T lymphocytes from these monkeys were assessed for reactivity with tetramers constructed with Mamu-A*01 and clade C-p41A or 89.6P-p41A (Fig. (Fig.7,7, four left panels). In monkey KPA, approximately half of the vaccine-induced CD8+ T-lymphocyte population that recognized the clade C-p41A peptide in association with Mamu-A*01 also recognized the 89.6P-p41A peptide in association with Mamu-A*01. In monkey 414, none of the CD8+ T lymphocytes that recognized clade C-p41A recognized 89.6P-p41A. Therefore, while the restricting MHC class I allele for the p41A peptide was shared by monkeys KPA and 414 and the immunization regimen was identical for these two monkeys, the cross-reactivity for a variant epitope peptide differed in these vaccinated animals. Importantly, these divergent patterns of CD8+ T-lymphocyte binding to tetramers constructed with variant p41A peptides were consistent with the functional T-lymphocyte responses elicited by the peptides. PBMC from monkey KPA generated an IFN-γ SFC response after in vitro exposure to clade C-p41A and a lower response to HxB2-p41A. PBMC from monkey 414 generated an IFN-γ SFC response after in vitro exposure to clade C-p41A but no response to HxB2-p41A (Fig. (Fig.5B5B).

FIG. 7.
Cross-reactivity of p41A epitope-specific CD8+ T-lymphocyte populations in clade C Env-immunized Mamu-A*01+ rhesus monkeys following vaccination and challenge. PBMC were isolated from monkeys KPA and 414 at week 27 following vaccination ...

Following challenge with SHIV-89.6P, changes were observed in the CD8+ T-lymphocyte populations that recognized p41A peptides in these two monkeys. As seen following vaccination alone, a fraction of the CD8+ T lymphocytes that bound the clade C-p41A tetramer also bound the 89.6P-p41A tetramer postchallenge in monkey KPA (Fig. (Fig.7,7, four right panels). However, in monkey 414, no CD8+ T-lymphocyte populations expanded that recognized both clade C-p41A and 89.6P-p41A. Importantly, in both of these monkeys, new CD8+ T-lymphocyte responses emerged following SHIV-89.6P challenge that had not been primed by prior vaccination—CD8+ T-lymphocyte populations that recognized 89.6P-p41A but not clade C-p41A (Fig. (Fig.7,7, four right panels).

The pattern of Vβ genes employed by these restricted populations of CD8+ T lymphocytes was, for the most part, consistent with these functional observations (Fig. (Fig.8).8). In monkey KPA, a population of CD8+ T lymphocytes employing Vβ 7B for p41A recognition mediated cross-recognition of clade C-p41A and 89.6P-p41A. However, a population of CD8+ T lymphocytes expressing Vβ 21 recognized clade C-p41A but not 89.6P-p41A in this monkey. Moreover, de novo-generated CD8+ T-lymphocyte populations were detected in this animal following SHIV-89.6P challenge, employing Vβ 13C.2, 13C.3, 15, and 16, that recognized 89.6P-p41A but not clade C-p41A. In monkey 414, vaccination generated populations of clade C-p41-specific CD8+ T lymphocytes that predominantly employed Vβ 13C.2, with very small contributions from Vβ 5B.1, 5B.1v, and 19. None of these populations cross-reacted with the 89.6P-p41A peptide. Following SHIV-89.6P challenge, these same Vβ genes were represented in the repertoire, but additional genes were also employed by the p41A-reactive CD8+ T lymphocytes, including 9A, 12.3, and 13A. These three Vβ gene families were all employed for 89.6P-p41A recognition. Interestingly, the data on the use of Vβ 13C.2 suggested that some cross-reactivity should have been observed at the CD8+ T-lymphocyte level between clade C-p41A and 89.6P-p41A. However, such cross-reactivity was not detected by tetramer staining (Fig. (Fig.77).

FIG. 8.
Vβ repertoire analysis of p41A epitope-specific CD8+ T-lymphocyte populations in clade C Env-immunized Mamu-A*01+ rhesus monkeys following vaccination and challenge. PBMC were isolated from monkeys KPA and 414 at week 27 ...

To explore further the absence of reactivity of CD8+ T lymphocytes from monkey 414 for both clade C-p41A and 89.6P-p41A, we determined the precise clones employed for peptide epitope recognition through use of CDR3 sequencing (Table (Table2).2). As a control, PBMC from monkey KPA were stimulated in vitro with either the clade C-p41A or the 89.6P-p41A peptide, and the Vβ 7B and Cβ primers were used to generate the CDR3 sequence from each of the cell populations. Consistent with the cross-reactivity of the CD8+ T lymphocytes for clade C-p41A and 89.6P-p41A, the same clonal population of CD8+ T lymphocytes mediated this cross-reactive recognition. Importantly, the sequencing of the Vβ 13C.2 cDNA from monkey 414 demonstrated that the clade C-p41A-specific CD8+ T lymphocytes and the 89.6P-specific CD8+ T lymphocytes in fact represented different clonal populations of T lymphocytes, even though they employed the same Vβ family gene to mediate this recognition. Therefore, these sequencing data were consistent with the demonstration of a lack of cross-reactivity as determined by tetramer staining.

TABLE 2.
Analysis of CDR3 sequences in clade C-p41A and 89.6P-p41A epitope-specific CD8+ T-lymphocyte populations in Mamu-A*01+ rhesus monkeys vaccinated with clade C Env and challenged with SHIV-89.6Pe

DISCUSSION

The experimental protocol employed in this study was configured to reflect the in vivo biology of a primed CD8+ T-lymphocyte population contacting viral antigen, with clonal constituents of that T-cell population then expanding in response to antigen recognition. The in vitro exposure of PBMC to epitope peptide is meant to reflect the in vivo exposure of PBMC to replicating virus. This exposure to peptide triggers the expansion of the subpopulation of primed CD8+ T lymphocytes that recognize that particular epitope peptide in association with MHC class I. Importantly, we have previously shown that such an in vitro peptide stimulation with the consequent expansion of a peptide/MHC class I-specific population of CD8+ T lymphocytes does not skew the TCR repertoire of the cell subpopulation being evaluated (20). Therefore, this experimental approach should yield biologically relevant data.

We chose to study the Mamu-A*01-restricted p41A epitope of HIV-1 Env as a representative epitope of a viral protein. It is an epitope that is predictably recognized by CD8+ T lymphocytes of Mamu-A*01+ rhesus monkeys that are either vaccinated with HIV-1 Env or infected by a SHIV construct. Importantly, it is not an overwhelmingly dominant epitope like the Mamu-A*01-restricted Gag p11C or Tat TL8, nor is it an epitope like the Mamu-A*01-restricted Pol p68A that elicits sporadic, very low frequency CD8+ T-lymphocyte responses (1, 3, 8, 28, 32). Finally, since no subtypes of Mamu-A*01 have been defined in Indian-origin rhesus monkeys, we can be confident that divergent T-lymphocyte responses reflect TCR rather than MHC class I-determined cellular interactions. Therefore, we felt that an analysis of CD8+ T-lymphocyte responses to this epitope should provide an important window through which to view the biology of CD8+ T-lymphocyte recognition of variant epitope peptides.

Our intent in the present study was to marry a number of methodologies to determine precisely which clonal populations of CD8+ T lymphocytes take part in the recognition of variant epitope peptides. Studying CD8+ T-lymphocyte binding to a series of tetramers constructed with the same MHC class I molecule and differing peptides provides a rapid assay for evaluating CD8+ T-lymphocyte recognition of variant Env sequences. To complement that data, we also employed a Vβ repertoire analysis to document which gene families are taking part in the peptide/MHC class I recognition. We felt that the use of a Vβ repertoire analysis provides data that complement the findings of Vβ gene CDR3 sequencing alone (2, 13, 33). When we wanted to clarify whether recognition of variant peptides mediated by CD8+ T lymphocytes expressing a single Vβ gene represented a single cross-reactive clone or multiple clones that employed the same Vβ gene, we performed CDR3 sequencing. These methodologies, when used in combination, provide a comprehensive definition of which CD8+ T-lymphocyte clones recognize a series of mutant forms of an epitope and which recognize a restricted population of potential epitopes.

Although the number of monkeys that we studied was small, the application of these methodologies for evaluating CD8+ T-lymphocyte populations in vaccinated and infected rhesus monkeys yielded findings that are troubling with regard to harnessing the immune system for the control of HIV-1. We found in a pair of rhesus monkeys with a shared MHC class I molecule that restricts p41A-specific CD8+ T lymphocytes that one of these animals could generate a population of cross-reactive p41-specific CD8+ T lymphocytes and the other could not. This divergence in responses was seen following vaccination and following infection. We were able to demonstrate that these differences reflect the use of distinct clonal populations of CD8+ T lymphocytes that mediate p41A recognition. Thus, one of these monkeys had a vaccine-elicited population of CD8+ T lymphocytes that could recognize a divergent infecting virus while the other monkey did not. This observation underscores the challenges that we face as we attempt to harness the immune response to control the spread of HIV-1.

Acknowledgments

We are grateful to Michelle Lifton for excellent technical assistance with flow cytometry, and we thank Lauren Dorosh for help with flow cytometry analysis.

This work was supported by the National Institute of Allergy and Infectious Diseases Center for HIV/AIDS Vaccine Immunology (grant AI067854 to N.L.L.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 July 2009.

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