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The TCR Repertoire of an Immunodominant CD8⁺ T Lymphocyte Population¹

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215

	Abstract

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The TCR repertoire of an epitope-specific CD8⁺ T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8⁺ T cell population that recognizes a dominant epitope of the AIDS virus, the CD8⁺ T cells recognizing the tetrameric Mamu-A*01/p11C_,CM complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01⁺ rhesus monkeys. This CD8⁺ T cell population exhibited selected usage of TCR V families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8⁺ T cell response was clearly polyclonal, a dominance of selected V⁺ cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V⁺ cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V⁺ cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V⁺ cell subpopulations and clones and suggests that dominant V⁺ cell subpopulations and clones can either be stable or evolve during a chronic infection.

	Introduction

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The CD8⁺ CTL play an important role in the immune containment of HIV-1 and SIV replication. The emergence of virus-specific CTL during primary HIV-1 and SIV infection is associated with a reduction in early viremia (1, 2, 3, 4). Moreover, high-frequency virus-specific CTL responses appear to contribute to a decrease in virus load and a delay in disease progression in chronically HIV-1-infected persons (5, 6, 7, 8). In fact, monoclonal anti-CD8 Ab-mediated depletion of CD8⁺ T lymphocytes in SIV-infected monkeys was associated with a marked increase in viral load (4, 9). In view of the importance of virus-specific CTL in AIDS immunopathogenesis, it is imperative that we understand this immune response at a cellular and molecular level.

Although virus-specific CTL have been extensively studied in the setting of HIV-1 infections, the TCR repertoire of CD8⁺ T cell responses specific for individual viral epitopes remains poorly characterized in infected individuals. To date, TCR repertoires of epitope-specific CTL have been studied by analyzing either the entire CD8⁺ T cell population or a limited number of CTL clones isolated from PBL of infected individuals (2, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Therefore, those studies have provided limited information concerning the molecular evolution of epitope-specific CD8⁺ T cells. Further studies of CD8⁺ T cell populations that recognize specific peptide/MHC class I complexes are needed to characterize the TCR repertoire of these epitope-specific CD8⁺ T lymphocytes.

The recent development of soluble tetrameric MHC class I/peptide complexes has allowed us to visualize and isolate epitope-specific CD8⁺ T cell populations from HIV-1-infected humans and SIV-infected monkeys (20, 21). This technology has facilitated an in-depth analysis of rhesus monkey CD8⁺ CTL specific for the dominant SIV Gag epitope p11C_,C-M presented to T lymphocytes by the MHC class I allele Mamu-A*01. In a previous study, we have assessed TCR gene expression in a series of CTL clones that recognize this SIV Gag epitope (10). In the present study, we have used the tetrameric Mamu-A*01/p11C_,C-M complex to characterize the TCR repertoire of this epitope-specific CD8⁺ T cell population and define the clonality of this immune response.

	Materials and Methods

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Animals and viruses

Rhesus monkeys (Macaca mulatta) were used in these studies. These animals were maintained in accordance with the guidelines of the Committee on Animals for Harvard Medical School and the "Guide for the Care and Use of Laboratory Animals" (National Academy Press, 1996). The rhesus monkeys were inoculated i.v. with SIV of macaques (SIVmac)³251 as described (21). All rhesus monkeys used in this study were Mamu-A*01⁺ as determined both by PCR-based MHC class I typing and by functional CTL assays as described previously (21).

Staining and sorting of CD8⁺ T cell subsets

Soluble tetrameric Mamu-A*01/p11C_,C-M complex was made as previously described (21). The tetramer was produced by mixing biotinylated Mamu-A*01/p11C_,C-M complex with PE-labeled streptavidin (Prozyme, San Leandro, CA) at a molar ratio of 4:1. The PE-coupled tetrameric Mamu-A*01/p11C_,C-M complex was used in conjunction with anti-CD8 (Leu2a)-FITC (Becton Dickinson, San Jose, CA), anti-CD8 (2ST8–5H7)-ECD (Beckman Coulter, Miami, FL) and anti-rhesus monkey CD3(FN18) directly coupled to APC. PE-coupled tetrameric Mamu-A*01/p11C_,C-M complex was used in conjunction with the directly labeled mAbs to stain either PBMC or single cells from lymph nodes or spleen isolated by density-gradient centrifugation over Ficoll-Hypaque. Sorting of potentially biohazardous specimens was performed on a Coulter EPICS Elite ESP (Beckman Coulter) located in a dedicated BSL-3 area. The sorter was set electronically to achieve enrichments of selected cell subsets of >99%. As a control study, the frequency of the tetramer-staining CD8⁺ T cells in uninfected animals also was determined. In our accumulating studies, CD8⁺ T cells recognizing tetrameric Mamu-A*01/p11C_,CM were either undetectable or constituted <0.1% of CD8⁺ T cells in naive Mamu-A*01⁺ macaques.

RNA extraction and cDNA synthesis

RNA was extracted from p11C_,C-M/Mamu*01 tetramer binding or nonbinding CD8⁺ T cell populations according to the instructions of the RNA extraction kit from Qiagen (Valencia, CA). cDNA was synthesized by using a method based on the switching mechanism at the 5' end of the RNA template (22, 23). The protocol was modified from the instructions in the SMART cDNA Synthesis Kit from Clontech Laboratories (Palo Alto, CA). Briefly, the cDNA reaction was catalyzed by using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD), the SMART primer provided in the Clontech Kit and a modified cDNA synthesis primer. The sequence for the modified cDNA synthesis primer was 5'-AAGCAGTGGTAACAACGCAGAGTACT₍₃₀₎NV-3' (N = A, C,G, or T; V = A, G, or C.). The double-stranded cDNA was made by a 10-cycle PCR with the primer and reagents provided in the SMART cDNA synthesis kit from Clontech Laboratories. In control experiments to validate the use of SMART cDNA synthesis in the present studies, SMART cDNA, cDNA that was synthesized by conventional methods, and cDNA derived from anchored PCR were all assessed for V gene expression (17, 24). Similar expression patterns of V gene families were seen in these different cDNA samples (data not shown).

TCR V family expression

PCR-based quantitation of V family expression was undertaken as described previously (25). Briefly, cDNA derived from each sample was aliquoted into 25 tubes, each containing a sense V family-specific and an antisense C-specific primer. As an internal control, each reaction tube also contained a pair of primers that amplified a 105-bp fragment of the constant region of macaque TCR -chain. The PCR was performed for 28 cycles, with each cycle at 95°C for 30 s, 60°C for 1 min, and 72°C for 2 min. The radiolabeled PCR products were electrophoresed through a 5% polyacrylamide gel, dried, and exposed to x-ray film. The separated V-C and C-C bands were measured for the radioactivity with an Ambis 100 (Ambis, San Diego, CA) or for density by a GS-700 Densitometer (Bio-Lab, Richmond, CA). To confirm the results of V family expression, the identified V families in each cDNA sample were assessed for complementarity-determining region 3 (CDR3) profiles through Genescan-based spectrotyping.

TCR CDR3 profile analysis

CDR3 profiles were analyzed by Genescan-based spectrotyping (26). cDNAs were amplified by PCR for expression of 24 V families by using individual V-specific primers and a C-specific primer as described previously (25, 26, 27). The second round of PCR was performed with nested V primers and a C primer, designed as described (26). The internal C primer was labeled at its 5' end with the Fam fluorophore (Applied Biosystems, Foster City, CA). The first-round PCR products were amplified for 15 cycles under the following conditions: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. One microliter of each reaction product was mixed with deionized formamide and a ROCK-500 size standard and then electrophoresed on a 5% acrylamide gel on a 377 DNA sequencer (Applied Biosystems). Data were analyzed for size and fluorescence intensity by using the Genescan software (Applied Biosystems). Experiments performed on samples obtained at three different times from four normal monkeys indicated that these CDR3 length analyses were highly reproducible and V-specific. Further cloning and sequencing in conjunction with the CDR3 length display allowed the prediction of CDR3 lengths. These lengths were expressed as predicted numbers of amino acids.

Sequencing and frequency analyses of TCR cDNA clones isolated from the tetramer-binding CD8⁺ T cells

The nonselective cloning and sequencing of TCR cDNA were done by using a technique modified from the anchored PCR, as described previously (24). The TCR cDNA was isolated from the SMART cDNA with the SMART primer (Clontech Laboratories) containing an EcoRI site and a C primer containing an XbaI site. The PCR was performed for 30 cycles under the following conditions: 95°C for 30 s, 60°C for 1 min, and 72°C for 2 min. The PCR products were digested with EcoRI and XbaI and ligated into the pSP65 plasmid (Promega, Madison, WI) for cloning and sequencing (2). For frequency analyses, 80–120 clones were sequenced and analyzed for each cDNA sample. The frequency of the individual clones was determined based on the percentage of each clone in the total clones isolated from each cDNA sample.

	Results

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Limited diversity of the TCR V repertoire in CD8⁺ T cell populations that recognize the tetrameric Mamu-A*01/p11C_,CM complex

To directly examine the TCR repertoire of a single viral epitope-specific CD8⁺ T cell population during an AIDS virus infection, tetrameric Mamu-A*01/p11C_,C-M complex-binding CD8⁺ T cell populations from SIVmac-infected, Mamu-A*01⁺ rhesus monkeys were isolated and assessed for their expression of 24 V gene families. The CD8⁺ T cell populations recognizing this tetrameric Mamu-A*01/p11C_,C-M complex were detectable in PBL of acutely and chronically SIVmac-infected monkeys (Fig. 1). The tetrameric Mamu-A*01/p11C_,CM complex-bound CD8⁺ T cells were isolated by flow cytometric sorting and assessed for their TCR V repertoire. A limited diversity of TCR V gene usage was seen in the tetrameric Mamu-A*01/p11C_,C-M complex-bound CD8⁺ T cell populations. The tetrameric Mamu-A*01/p11C_,CM complex-bound CD8⁺ T cell populations predominantely expressed TCR with certain V gene family members (Fig. 2). Interestingly, a V13-expressing cell subpopulation was consistently dominant in the tetrameric Mamu-A*01/p11C_,C-M complex-bound CD8⁺ T cell populations from the monkeys examined. The V13-expressing cell populations constituted from 17 to 48% of the tetramer⁺ CD8⁺ T cell populations sorted from the PBL of the SIVmac-infected monkeys (Fig. 2). In contrast, the CD8⁺ T cell populations derived from the PBL of the same monkeys that did not bind to tetrameric Mamu-A*01/p11C_,CM complex exhibited a diverse TCR V repertoire. All 24 V families were expressed in these tetramer-negative cell populations (Fig. 2). Therefore, these results suggested that the tetrameric Mamu-A*01/p11C_,C-M complex interacted with only selected CD8⁺ T cell subpopulations.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Detectable CD8+ T cell populations that recognize the tetrameric Mamu-A*01/p11C,C-M complex in SIVmac-infected, Mamu-A*01+ rhesus monkeys. Data were derived from the flow cytometric analysis and showed the percentage of tetramer+ cells in CD8+ T cells at the time the cells were sorted for molecular analysis.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Limited diversity of the TCR V repertoire of the CD8+ T cell population that recognizes the Mamu-A*01/p11C,C-M complex. The data shown on the left were derived from the CD8+ T cells that bound to the tetrameric Mamu-A*01/p11C,C-M complex (tetramer+), whereas those on the right were derived from the cells that did not bind the tetramer (tetramer-). These CD8+ T cell populations were isolated by sorting PBL obtained 2 wk (from two monkeys, Mm8898 and Mm8798) and 2–5 years (from three other animals) after SIVmac infection. The representation of individual V families in a lymphocyte population are shown as a percentage of the expression of all of the 24 families (25 ).

Restricted CDR3 profiles in the V⁺CD8⁺ T cell populations that recognize tetrameric Mamu-A*01/p11C_,CM complex

To determine whether the CD8⁺ T cells that recognize this single SIV Gag epitope are restricted in the CDR3 regions they use, we examined the CDR3 profiles of selected V-expressing cell subpopulations in the tetrameric Mamu-A*01/p11C_,C-M complex-bound CD8⁺ T cell population. CDR3 spectrotyping analysis revealed a restricted pattern of CDR3 profiles in the selected V-expressing CD8⁺ T cell subpopulations that recognize the tetrameric Mamu-A*01/p11C_,C-M complex. A CDR3 of a single length was identified in the majority of these selected V-expressing CD8⁺ T cell subpopulations, whereas the CD8⁺ T cells that did not bind the tetramer displayed multiple CDR3 lengths (Fig. 3). A CDR3 length of 11 aa appeared to be the most frequently used by the selected V⁺CD8⁺ T cell subpopulations in the tetramer-bound lymphocytes (Fig. 3). These results extend the studies of V family expression described above and demonstrate at a CDR3 level the limited diversity of TCR repertoire in the CD8⁺ T cell subpopulations that bind to the tetrameric Mamu-A*01/p11C_,C-M complex.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Restricted CDR3 profiles in the selected V+CD8+ T cell subpopulations that recognize the tetrameric Mamu-A*01/p11C,C-M complex. Shown are histograms generated in the spectrotyping analysis of CDR3 lengths used by selected V+ subpopulations of the tetramer-positive (tetramer+) and -negative (tetramer-) CD8+ T cell populations. Monkey designations are noted at the top of this figure. Fragment lengths in nucleotide numbers are displayed on the x-axis and fluorescence intensity on the y-axis. The number of nucleotides in the different CDR3 lengths were determined in control experiments (see Materials and Methods) and are expressed as predicted number of amino acids. The short line at the bottom of each histogram represents the CDR3 length of 10 aa. Shown are only those histograms of V families in which CDR3 profiles were identified in the cDNA from the purified tetramer+ CD8+ cells and the corresponding V families from the control tetramer- CD8+ cells. The infection status of the animals and the time following infection at which tetramer+ and tetramer- CD8+ T cells were obtained are the same as described in the legend of Fig. 2.

Clonal dominance in the V⁺CD8⁺ T cell populations that recognize tetrameric Mamu-A*01/p11C_,CM complex

As a complementary study, sequencing of TCR cDNA was used to characterize the TCR repertoire and clonality of the CD8⁺ T cell population that binds to the tetrameric Mamu-A*01/p11C_,C-M complex. These sequence analyses yielded results similar to those shown in the PCR-based quantitation of V gene family expression and the CDR3 profile studies. A predominant use of the sellected V gene families was seen at the sequence level in the CD8⁺ T cell population that bound to the tetrameric Mamu-A*01/p11C_,C-M complex (Figs. 2 and 4, A and B). These selected V⁺CD8⁺ T cell subpopulations exhibited monoclonal or oligoclonal dominance and restricted CDR3 lengths. Nevertheless, the CD8⁺ T cell populations recognizing the tetrameric Mamu-A*01/p11C_,C-M complex were clearly polyclonal (Fig. 4). Furthermore, the number of dominant clones in the CD8⁺ T cell populations that bound the tetrameric Mamu-A*01/p11C_,C-M complex appeared to be smaller during the chronic than during the acute phase of SIVmac infection (Fig. 4, A and B). These results provide further evidence that SIVmac-infected Mamu A*01⁺ rhesus monkeys develop a p11C-specific CD8⁺ T cell response comprised of limited numbers of V-expressing cell subpopulations.

View larger version (83K): [in this window] [in a new window]   FIGURE 4. Clonal dominance in the V repertoires of the CD8+ T cell populations recognizing the Mamu-A*01/p11C,C-M tetramer complex. A nonselective cloning strategy for TCR sequences was used (see Materials and Methods) with the cDNA prepared from the tetramer+ CD8+ T cells isolated through cell sorting. The frequency of clonatypic sequences is expressed as the percentage of that clone among the total clones identified in the cDNA from the tetramer+ CD8+ T cells.

Dominant V⁺ cell subpopulations or clones in the p11C-specific CD8⁺ T cell population can change during the course of SIVmac infections

Finally, we sought to examine the temporal evolution of the TCR repertoire of the CD8⁺ T cell populations that recognize the tetrameric Mamu-A*01/p11C_,C-M complex. Cells that bound to the tetrameric Mamu-A*01/p11C_,C-M complex were isolated by sorting PBL of two Mamu-A*01⁺ rhesus monkeys 0.5, 2, and 7 mo after SIVmac infection (Fig. 5). Although some selected V⁺CD8⁺ T cell subpopulations maintained their clonal dominance over time, others fluctuated in their clonal representation in the TCR repertoire. These varied patterns of clonal evolution were seen at both the level of V family expression and CDR3 profiles during the 7-mo follow-up of the SIVmac-infected monkeys (Figs. 6 and 7). Some of the clones identified in PBL during the period of acute infection maintained their dominance in the repertoire of the tetramer-binding CD8⁺ T cell populations, whereas others were either lost or decreased in the frequency of their representation within this cell population (Fig. 8). Interestingly, those clones that lost their dominance over time appeared to be replaced by newly evolving clones that became dominant during chronic SIVmac infection (Fig. 8). Therefore, these results provide molecular evidence that dominant V⁺ cell subpopulations or clones in the repertoire of the p11C-specific CD8⁺ T cell population can be stable or changing during the course of SIVmac infections.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. CD8+ T cell populations that recognize the tetrameric Mamu-A*01/p11C,C-M complex were detectable over time after SIVmac infection in the monkeys. The legend is the same as that to Fig. 1.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Evolution of tetramer-binding CD8+ T cell subpopulations. Shown was the analysis of the V family expression in the tetramer-binding CD8+ T cell populations obtained from PBL of two monkeys 0.5, 2, and 7 mo after infection.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Evolution of the CDR3 profiles of tetramer-binding CD8+ T cell population. Shown is the analysis of the CDR3 profiles of selected V-expressing cell subpopulations in the tetramer-binding CD8+ T cell population obtained from PBL of two monkeys 0.5, 2, and 7 mo after infection.

View larger version (47K): [in this window] [in a new window]   FIGURE 8. Evolution of dominant clones in tetramer-binding CD8+ T cell population. Shown is the frequency analyses of particular TCR clones. These analyses show that the dominant clones in the tetramer-binding CD8+ T cell population can be either stable or replaced during the course of SIVmac infections. Sequencing and frequency analyses were performed as described in the legend to Fig. 4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The patterns of V dominance in the Mamu-A*01/p11C_,C-M tetramer-binding CD8⁺ T cell populations varied among individual SIVmac-infected monkeys. The differences among the monkeys is unlikely to be a result of biases introduced by the molecular analyses themselves, because differing patterns of V family usage were confirmed by PCR amplification with primers that encompass all of the identified V families and known clonotypic sequences (see Materials and Methods; data not shown). Moreover, variability in the patterns of dominance of selected V families in different monkeys also was seen in our previous study of epitope-specific CTL clones (10, 24). This variability also cannot be explained by the existence of multiple subtypes of the Mamu-A*01 allele, where each subtype might select for a different V⁺CD8⁺ T cell subpopulation, because sequencing of the 2 domain of the Mamu-A*01 allele from >600 animals has demonstrated no polymorphism in that gene in Indian rhesus moneys (data not shown). It is likely that the variation in V dominance reflects the differences in thymic selection driven by the complexity of MHC haplotypes in outbred species (28). We also cannot exclude the possibility that differences in cytokine profiles among the infected monkeys may contribute to the distinct V patterns seen in individual animals (29).

The present study provides molecular evidence that some of the dominant V⁺ cell subpopulations or clones in the p11C-specific CD8⁺ T cell population can be stable over time during the course of SIVmac infections. These results are consistent with a recent report describing the persistence of a peptide epitope-specific CTL clone in an HIV-1-infected person (11, 30, 31). The stability of at least a portion of the repertoire of SIVmac p11C-specific CD8⁺ T cell populations suggests that viral peptide epitope-specific CD8⁺ T cell clones can continuously proliferate and expand in AIDS virus-infected individuals. Such prolonged expansions of dominant clones may provide an advantage in containing persistent viral replication in infected individuals.

Others of the dominant V⁺ cell subpopulations or clones in the p11C-specific CD8⁺ T cell population appear to vary during the course of SIVmac infections. These changes in the TCR repertoire do not appear to be driven by the emergence of viral mutants, as we were unable to identify a high frequency of mutations in the epitope-coding region of SIVmac gag during the 7-mo follow-up of monkeys 9398 and 9498 (data not shown). Rather, the evolving clonal dominance in the TCR repertoire over time may be driven by host factors, some of which may be consequences of the SIV infection itself. Thus, for example, a burst of viral replication and an associated marked depletion of CD4⁺ T cells occur during the period of primary SIVmac infection in monkeys. This high viral load and sharp decline in CD4⁺ T helper cells may affect the proliferation or turn-over of specific CD8⁺ T lymphocyte clones. These viral and immune changes may, in turn, affect the TCR repertoire of the CD8⁺ virus-specific CTL. As a result, some clones that are present during primary infection may be replaced by others that emerge during the chronic phase of infection. It is also possible that changes in production of specific cytokines as described above may select for dominant clones in the TCR repertoire during SIV infection of a monkey. Finally, we cannot exclude the possibility that random selection may underlie the changes in clonal dominance in the TCR repertoire of the CD8⁺ T cell population recognizing the Mamu-A*01/p11C_,C-M complex. By whatever mechanism, the results of the present study suggest that some V⁺ populations or clones that emerge in the chronically infected monkey can replace previously dominant cell subpopulations in the repertoire of the p11C-specific CD8⁺ T cell response during SIVmac infections.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants ROI HL64560 (to Z.W.C.) and AI20729 (to N.L.L.).

² Address correspondence and reprint requests to Dr. Zheng W. Chen, 330 Brookline Avenue, RE113, Boston, MA 02215.

³ Abbreviations used in this paper: SIVmac, SIV of macaques; CDR3, complementarity-determining region 3.

Received for publication November 7, 2000. Accepted for publication January 19, 2001.

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