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Analysis of the TCR Variable Gene Repertoire in Chimpanzees: Identification of Functional Homologs to Human Pseudogenes¹

Dirk Meyer-Olson^*, Kristen W. Brady^*, Jason T. Blackard, Todd M. Allen^*, Sabina Islam^*, Naglaa H. Shoukry, Kelly Hartman^*, Christopher M. Walker and Spyros A. Kalams^2,*,

^* Partners AIDS Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02129; Gastrointestinal Unit 3, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114; Division of Molecular Medicine, Children’s Research Institute, Columbus, OH 43205; and Infectious Diseases Unit, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232

	Abstract

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Chimpanzees are used for a variety of disease models such as hepatitis C virus (HCV) infection, where Ag-specific T cells are thought to be critical for resolution of infection. The variable segments of the TCR genes are polymorphic and contain putative binding sites for MHC class I and II molecules. In this study, we performed a comprehensive analysis of genes that comprise the TCR variable gene (TCRBV) repertoire of the common chimpanzee Pan troglodytes. We identified 42 P. troglodytes TCRBV sequences representative of 25 known human TCRBV families. BV5, BV6, and BV7 are multigene TCRBV families in humans and homologs of most family members were found in the chimpanzee TCRBV repertoire. Some of the chimpanzee TCRBV sequences were identical with their human counterparts at the amino acid level. Notably four successfully rearranged TCRBV sequences in the chimpanzees corresponded to human pseudogenes. One of these TCR sequences was used by a cell line directed against a viral CTL epitope in an HCV-infected animal indicating the functionality of this V region in the context of immune defense against pathogens. These data indicate that some TCRBV genes maintained in the chimpanzee have been lost in humans within a brief evolutionary time frame despite remarkable conservation of the chimpanzee and human TCRBV repertoires. Our results predict that the diversity of TCR clonotypes responding to pathogens like HCV will be very similar in both species and will facilitate a molecular dissection of the immune response in chimpanzee models of human diseases.

	Introduction

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Humans and chimpanzees, which separated from a common ancestor more than five million years ago (1, 2, 3), share 98.6% identity between their genomes (4). This high degree of homology, coupled with a susceptibility to HCV infection, make chimpanzees the most important disease model for hepatitis C virus (HCV)³ infection. Recent studies in chimpanzees and humans have shown that broadly reactive HCV-specific T cell responses established early after infection are associated with clearance of infection (5, 6, 7, 8) and that establishment of chronic infection is associated with rapid viral escape from CTL recognition (9).

T cell recognition is mediated by the T cell receptor (TCR), which recognizes (viral) Ags presented by MHC class I or II complexes. TCR diversity is generated by the rearrangement of V, D (for the TCR chain), J, and C regions. The random insertion of nongermline-encoded nucleotides at the junctions of these rearranged segments provides additional diversity and is the main site of Ag recognition (complementarity determining region (CDR)3). The most polymorphic region within the TCR chain is the TCR V region (TCRBV), which encodes the CDR1 and CDR2 site. These sites within the V regions contribute to the recognition of the peptide/MHC complex mainly by interacting with the MHC molecule. The potential human TCRBV repertoire consists of 40–48 functional TCRBV genes belonging to 21–23 families based on DNA sequence similarities (10, 11).

Diversity of the TCR repertoire has been hypothesized to be associated with successful immune responses against various pathogens (12, 13, 14). Characterization of the TCRBV repertoire in chimpanzees is incomplete, so it is not possible to study how diversity in this gene family influences the host-pathogen relationship. In general, genes encoding MHC molecules are highly polymorphic and evolve rapidly (15, 16). Evolutionary pressures are not neutral but instead are probably driven by exposure to pathogens specific to each species. The orthologous class I MHC A, B, and C genes of humans and chimpanzees are highly polymorphic but have no species-defining characteristics. Nevertheless, no common alleles are shared between these two closely related species and recent data indicate a substantial reduction in the MHC class I repertoire in chimpanzees (17). To date, alleles representative of some common HLA lineages such as HLA-A2 have not been found in chimpanzees even though recognition of peptides bearing the HLA-A2 supermotif in hepatitis B-infected animals has been demonstrated (18). It might be predicted that this rapid pace of evolution also contributes to diversification of ligands that bind MHC molecules. Killing-inhibitory receptors (KIR) that regulate NK cell activity by binding classical MHC class I A and B molecules have also evolved rapidly after speciation. Indeed, the rate of KIR gene evolution between humans and chimpanzees is comparable to or even faster than that of class I MHC molecules themselves (19). Based on analysis of a small subset of V genes the TCRBV repertoires of humans and chimpanzees are predicted to be highly conserved (20, 21, 22). However, for many human TCRBV families, no corresponding chimpanzee sequences have been described thus far.

The goal of this study was to compare the extent of TCRBV gene diversity in chimpanzees and humans after more than five million years of independent evolution. We performed a comprehensive analysis of over 700 cDNA sequences from chimpanzee PBMCs and T cell lines. Over the course of this study, we confirmed that chimpanzee TCRBV sequences were highly homologous to their human gene counterparts and were distributed among 25 human TCRBV families. We also demonstrate that several chimpanzee TCRBV genes, some derived from HCV-specific cell lines and clones, correspond to human TCRBV pseudogenes. Accumulation of nonfunctional TCRBV segments indicates ongoing evolution of these important immune response genes after separation of Pan troglodytes and Homo sapiens species.

	Materials and Methods

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Nomenclature

The nomenclature of P. troglodytes TCRBV genes is based on sequence similarity to their human homologues (23, 24). The nomenclature used in this manuscript is according to the suggestions of Rowen et al. (10) and LeFranc et al. (11). However, for the convenience of the readers, the older, but still widely used, nomenclature by Arden et al. (24) is included in parentheses. The numbering system of the amino acids is in accordance with the ImMunoGeneTics database system (http://imgt.cnusc.fr:8104).

All described P. troglodytes sequences have been submitted to GenBank (accession numbers AY168667-AY168708).

cDNA synthesis

After RNA was isolated from mononuclear cells (25) a modified anchored RT-PCR was performed as previously described (14, 26) using a gene-specific primer for the TCR C region (5'-AAT CCT TTC TCT TGA CCA TG-3') and an anchor primer (5'-AAGCAGTGGTAACAACGCAGAGTAGCGGG-3'; Clontech Laboratories, Palo Alto, CA). Three microliters of the isolated RNA was transcribed in a 10-µl reaction consisting of 5x first-strand buffer, 50x dNTP, and 20 µM DTT and Powerscript reverse transcriptase (Clontech Laboratories). After an annealing period of 10 min, the reaction was incubated at 42°C for 90 min and heat inactivated at 70°C. Amplification by PCR was performed using the TCR C region based primer 3'-TTG GGT GTG GGA GAT CTC TGC TTC TGA TGG-5' and the anchor-specific primer 5'-AAT CCT TTC TCT TGA CCA TG-3'. A PCR reaction was performed using 10x PCR buffer, 50x dNTP, and 50x AdvanTaq polymerase (all Clontech Laboratories). PCR conditions were 5 cycles at 94°C for 30 s and 72°C for 3 min; 5 cycles at 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min; and 30 cycles at 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min. PCR products of 500–600 bp were cloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and used to transform Escherichia coli. Selected colonies were sequenced using the Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems, Norwalk, CT) and capillary electrophoresis on an ABI 3700 PRISM automated sequencer (Applied Biosystems).

Phylogenetic analysis of TCRBV sequences

A consensus sequence from full-length V regions (200 bp) was generated using Sequencher (Gene Codes, Ann Arbor, MI). Data base searches were performed with GCG FASTA sequence analysis software (Genetics Computer Group, Madison, WI). Consensus sequences were aligned as described against a local TCR database with published sequences for the TCR V chain, which were derived from GenBank and European Molecular Biology Laboratory entries. Sequences were truncated after the leader sequences before TCRBV amino acid position 1 and at the beginning of the CDR3 region at position 106 (amino acid positions are defined according to the ImMunoGeneTics database numbering). Alignment was performed using the neighbor-joining method of ClustalW (27) and further phylogenetic analysis was performed using MEGA software (28).

The Jukes-Cantor estimate of the number of nucleotide substitutions per site was <0.05 between the human and chimpanzee TCRBV homologs. Therefore, we generated phylogenetic trees based on the p-distance using the neighbor-joining method (29), which gives us the lowest amount of variances as compared to other methods (30). Positions with deletions in the alignment were deleted during further analyses. The reliability of the branching order within each phylogenetic tree was confirmed by bootstrap analysis using 1000 replications (31). The branching order of the tree was further confirmed using the Interior Branch Test of the MEGA software. In addition, we generated minimal evolution trees and maximum parsimony trees to confirm the topology. Analysis rates of synonymous (dS) and nonsynonymous substitutions (dN) per site were performed with the Nei-Gojobori method of the MEGA software (28).

Generation of HCV-specific cell lines

HCV-specific T cell lines were derived either by stimulation with HCV-specific peptides or limited dilution as described earlier (9). Briefly, for the generation of HCV-specific T cell clones, peripheral blood cells or expanded liver cells were cloned by limited dilution and tested against either HCV peptide-pulsed B cell lines or B cell lines infected with vaccinia constructs containing the respective HCV sequence. HCV-specific T cell lines were generated by repeated stimulation with HCV peptides. HCV-specific T cells were purified using a commercial IFN- secretion assay (Miltenyi Biotec, Auburn, CA) as described previously (32). Briefly, 30 to 40 x 10⁶ T cells were incubated with 20 µM peptide and 1 µg/ml of the anti-CD28 and anti-CD49d mAbs (BD Biosciences, San Jose, CA) on six-well plates at 37°C, 5% CO₂ for 6–8 h. Cells were subsequently labeled with a bispecific CD45/IFN- Catch Reagent and incubated for 45 min at 37°C. After several washes, the IFN--producing cells were stained with a second IFN- detection Ab conjugated to PE and separated by cell sorting using the FACSVantage cell sorter (BD Biosciences).

	Results

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Detection and alignment of TCRBV regions derived from chimpanzees and humans

The characteristics of the animals in this study are outlined in Table I. Three of these animals are chronically infected with HCV and one animal has resolved infection, clearing HCV within 100 days after primary challenge.⁴ The immune responses and clinical course of these animals has been described in detail elsewhere (7, 9, 33, 34, 35, 36). We generated 796 cDNA clones corresponding to fully rearranged TCRBV regions. Sequences were derived after RNA extraction from peripheral blood cells and HCV-specific T cell lines as described in Materials and Methods. Human homologues were identified through database searches (ImMunoGeneTics database/National Center for Biotechnology Information) andthe corresponding chimpanzee TCR sequences were aligned and compared to known human TCRBV sequences. Fig. 1 givesan overview of the predicted amino acid sequences derived from Pan troglodytes aligned with their human TCRBV counterparts. We identified TCRBV sequences corresponding to 42 different human TCRBV genes. Of those, 38 correspond to functional human TCRBV genes. Thus, 79–90% of the potential human TCRBV repertoire (with an estimation of 40–42 functional human TCRBV genes) can also be found in chimpanzees.

View larger version (105K): [in this window] [in a new window]   FIGURE 1. Predicted amino acid alignment of P. troglodytes (Pt) and human (Hu) TCRBV sequences. Alignment was performed according to the suggestions of Lefranc et al. (11 ). For numbering of amino acids positions the IMGT unique numbering for the V regions was used. Framework (FR) and CDR of the TCRBV are indicated. CDR1 and CDR2 are boxed. Nomenclature of TCRBV sequences according to Rowen et al. (10 ). In brackets are the older but also commonly used nomenclature suggested by Arden et al. (24 ). Conserved amino acids are highlighted by black boxes. Differences between human and chimpanzee sequences are highlighted in yellow. "z" indicates a stop codon. Accession numbers for the human sequences are U66059, U66060, and U66061, respectively, with the exception of huBV29-1 (huBV4S1) (X04926), BV5-5 (huBV5S3) (X57611), huBV7-9 (huBV6S4A2T) (U03115), huBV7-7 (huBV6S6A2T) (X57607).

View larger version (111K): [in this window] [in a new window]   FIGURE 1A. Continued

Phylogenetic analysis (Fig. 2) of P. troglodytes TCRBV sequences demonstrate that we could identify chimpanzee cDNA molecular clones corresponding to 25 human TCRBV families (10). Single TCRBV genes are included into a family if they share >75% sequence identity (11, 24). All sequences of P. troglodytesshowed a close phylogenetic relationship to a distinct human counterpart. Therefore, we observed an alignment of closely related genes between the two species rather than an alignment within one species. Some human TCRBV families are multigenic, containing several related members. Homologues of these genes were also found in the chimpanzee, where we could identify five genes within the TCRBV5 (BV5) family, four within the TCRBV6 (BV13), and six within the TCRBV7 (BV6) family. Thus, the TCRBV repertoire in chimpanzee has a similar structure to that of humans.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Phylogenetic analysis of human and chimpanzee TCRBV sequences. A neighbor-joining tree demonstrates the association between P. troglodytes TCRBV genes and their respective human homologues. Only bootstrap values of >70% (based upon 1000 bootstraps) are shown. TCRBV families with more than three chimpanzee homologues are highlighted by brackets on the right.

Some genes showed 100% identity between Homo sapiens and P. troglodytes at the amino acid level (Fig. 1). If differences at the amino acid level between both species were noted, they were observed in the framework as well as CDR1 and CDR2 sites of the TCRBV gene (Table II). To investigate the relationship between human and chimpanzee TCRBV sequences, we calculated the number of synonymous and nonsynonymous mutations in the framework and CDR sites in human and chimpanzee TCRBV sequences (Table II). As expected, we found a high degree of homology in both regions between the different species. Confirming earlier observations (21), we found BV4-1 (BV7S1) to be identical between human and chimpanzees at the amino acid level. Moreover, we found no differences at the amino acid level between human and chimpanzee for BV7-2 (BV6S5), BV10-3 (BV12S1), BV24 (BV15S1), and BV30 (BV20S1). BV24 (BV15S1) shared identity at the nucleotide level between humans and chimpanzees within the coding region; however, there were nucleotide differences between both species within the leader sequence (data not shown).

Previous analyses of different sites within the TCR V region in humans and other species have demonstrated amino acid conservation within the framework region as compared to the CDR regions in the unselected TCRBV repertoire (37, 38). To analyze the TCRBV genes for selective pressure on the CDR and framework regions within the chimpanzee, the proportion of synonymous substitutions per site (pS) were plotted against nonsynonymous substitutions (pN) per site (39) in these regions. We observed that the majority of nucleotide substitutions over the length of the TCR were synonymous (comparison of pN vs pS; p < 0.001) (Fig. 3). A more detailed analysis of the relationship between nonsynonymous and synonymous mutations per site revealed a decreased ratio of nonsynonymous to synonymous mutations in the framework region compared to that of the CDR1 and CDR2 regions (0.702 vs 0.880; p < 0.001) (Fig. 3). This indicates selection against nucleotide substitutions leading to amino acid changes, or "purifying selection", within the framework regions (37, 38) and demonstrates a high degree of conservancy for amino acid residues necessary for determining the structure of the TCR complex.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Relationship of synonymous and nonsynonymous mutations per site within the TCRBV CDR 1 + 2 and framework sites. Rate of synonymous mutations per site (pS) and nonsynonymous mutations per site (pN) within the CDR 1 + 2 (A) and framework (B) sites of chimpanzee TCRBV. Each point represents a pairwise comparison between two chimpanzee TCRBV sequences. Analysis of pS and pN was performed using the Nei-Gojobori method as described in Materials and Methods.

To analyze the evolution of the TCRBV between humans and chimpanzees in more detail, we performed a comparison of synonymous (pS) and nonsynonymous (pN) mutations per site between distinct homologues of human and chimpanzee TCRBV sequences. Overall, there was a higher rate of synonymous mutations than nonsynonymous mutations when both species were compared over the entire length of the TCRBV sequences (Table II). However, a comparison of the framework regions and the CDR1 and CDR2 regions between species revealed that the framework regions had a lower frequency of both synonymous (0.019 vs 0.023; p < 0.01) and nonsynonymous (0.010 vs 0.014; p < 0.01) mutations when compared to the CDR1 and CDR2 regions, which indicates a higher degree of conservancy within framework regions of the TCR during evolution after the separation of human and chimpanzee lineages (Table II).

Sequence comparison of human TCRBV pseudogenes with functionally rearranged regions derived from chimpanzees

Although the data thus far have confirmed the high degree of homology between the TCRBV sequences of chimpanzees and humans, four of the detected genes derived from chimpanzees are not functionally expressed in humans.

BV5-7 (BV5S7). We could identify cDNA clones that showed a close alignment to the human BV5-7 (BV5S7) pseudogene (Figs. 1 and 2). The alignment of the predicted amino acid sequences of PtBV5-7 with the huBV5-7 is shown in Fig. 4A. In humans, a conserved tryptophan at amino position 41 "TGG" has mutated to a serine residue "TCG" due to a point mutation (10). The loss of the conserved tryptophan residue leads to an insufficient protein folding of the TCRBV chain. In humans, no functional rearrangement of the BV5S7 V region has been described to date (10).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Alignment of human pseudogenes with their corresponding chimpanzee sequences. Alignment of PtBV5-7 (BV5S7) (A), PtBV3-2 (BV9S2) (B), and PtBV21-1 (BV10S1) (C) with their respective human homologues. Nucleotide sequences and the predicted amino acid sequence are displayed. Amino acid positions are shown on top of the sequence. Regions with the mutations leading to the loss of functional expression of the human genes are boxed.

BV21-1 (BV10S1). The human BV21-1 (BV10S1) contains a frameshift within the leader sequence (10). Comparison of human and chimpanzee sequences helped to identify an insertion of a cytidine nucleotide at amino acid position -3 (Fig. 4C). One published sequence (21) from rhesus monkeys (U04575), which showed close alignment with human and chimp BV21-1 (BV10S1), did not contain the frameshift mutation found in humans.

BV23 (BV19S1). The human BV23 (BV19S1) pseudogene is rendered nonfunctional because of a point mutation in the 5'-splice region within the leader sequence (10). cDNA derived from humans encoding BV23 (BV19S1) therefore includes a 150-bp intron within the variable sequence of the V region. We were able to amplify a completely rearranged chimpanzee cDNA with high homology to the human BV19S1 that did not contain the intervening sequence. Although full confirmation of the absence of this point mutation would require sequencing the chimpanzee germline DNA of this region, the presence of the full-length properly rearranged cDNA strongly suggests that the splice region in the chimpanzee BV19S1 is still intact. Taken together, these data demonstrate there are at least four human pseudogenes that are functionally rearranged in chimpanzees, highlighting important differences between the human and the chimpanzee TCR repertoire.

Detection of functional homologs to human pseudogenes in HCV-specific T cell lines derived from HCV-infected chimpanzees

To determine whether intact chimpanzee TCR sequences corresponding to human TCR pseudogenes could be a part of the virus-specific T cell repertoire in chimpanzees, we performed an extensive screening of HCV-specific T cell lines and clones in chimpanzees. We analyzed 47 HCV-specific T cell lines, which were derived from four HCV-infected chimpanzees. These sequence data were included in our alignment of the chimpanzee T cell receptor repertoire (Figs. 1 and 2). We could detect a functional rearrangement of human pseudogenes in several HCV-specific CD8⁺ cell lines derived by limiting dilution cloning. In one chimpanzee (CH503) with chronic HCV infection, a T cell clone (10.D) using PtBV5-7 (BV5S7) was specific for an HCV-NS3 epitope (GDFDSVIDC) which was presented by Patr-B*1601 (41). Another cell line (369) was specific for an HCV-E1 epitope and was also restricted by PatrB1601 (GDASRCWVA). Five of eight TCR sequences from this HCV-specific cell line also showed a rearrangement of PtBV5-7 (BV5S7).

Two CD4⁺ T cell lines (1C and 4D) could be isolated from the peripheral blood 6 years after resolution of HCV infection in CB0572.⁴ Both cell lines were directed against an epitope located in NS5_2081–2090 ("ALWRVSAEEY"). TCRBV regions isolated from this cell line contained rearranged PtBV14 (BV16S1) (six of eight for 4D and seven of nine for 1C) and PtBV3-2 (BV9S2) (two of eight for 4D and two of nine for 1C). In both cell lines, the TCR sequences with the PtBV3-2 (BV9S2) and PtBV14 (BV16S1) genes were identical at the nucleotide level, indicating identical TCR clonotypes. The presence of two inframe TCR transcripts in independently derived CTL clones suggests these are dual TCR T cells, which has been described to occur in 1% of cases (42). These data suggest that human pseudogenes are not only functionally rearranged in the chimpanzee TCR repertoire but also contribute to the Ag-specific T cell response against HCV.

	Discussion

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The widespread use of chimpanzees as a model where T cell responses play a substantial role in disease pathogenesis gave the rationale for a comprehensive analysis of the chimpanzee TCRBV repertoire in comparison to humans. Understanding the similarities between chimpanzees and humans at the level of MHC and TCR genes will aid our understanding of the factors that govern control of chronic viral infections.

Previous work demonstrated a close phylogenetic relationship between some human and chimpanzee TCRAV (43) and TCRBV genes (20, 21). However, for a substantial proportion of human TCRBV genes, no chimpanzee homologues have yet been described. In this study, we describe TCRBV sequences derived from chimpanzee PBMC or cell lines which could be aligned with 42 different human TCRBV genes, and which correspond to 25 described human TCRBV families. All functionally rearranged human TCRBV families were represented in the chimpanzee TCRBV repertoire. We found no evidence that new TCRBV families might have evolved in the chimpanzee TCRBV repertoire. Moreover, we could detect a similar accumulation of genes within the human multigene TCRBV families TCRBV5 (BV5), TCRBV6 (BV7), and TCRBV7 (BV13). These data indicate a high degree of conservation of the TCRBV repertoire between humans and chimpanzees during evolution.

This is an important finding in the light of the rapid variations observed within MHC-I complex during that same evolutionary period (15, 16), which included the development and the loss of different MHC class I lineages between humans and chimpanzees. Another study investigating the TCRBV repertoire of the more distantly related cotton-top tamarin monkeys, which have a limited diversity of the MHC class I repertoire as compared to humans, described the evolution of new TCRBV genes as well as changes in the repertoire of TCRBV multigene families (37). Taken together, these and our findings indicate a substantial delay of changes within the TCRBV repertoire in response to evolutionary changes of the MHC-I complex. In contrast, studies of KIR genes, which interact with MHC class I Ags as well, did show substantial differences between humans and chimpanzees (19).

In addition to the highly similar structure of the TCRBV repertoire between species (i.e., the same number and similar size of TCRBV families) there was also a high degree of similarity between the individual genes, with a substantial proportion of genes having identical amino acid sequences as described before for the BV4-1 (BV7S1) gene (21). This high degree of phylogenetic relationship has led to the conclusion that the TCR repertoire as observed in humans and chimpanzees might be highly optimized, leaving little room for extensive genetic variation (20, 21, 22). This was also confirmed by separate investigation of the TCR framework and CDR1 + 2 regions within each species that demonstrated identical patterns of nonsynonymous and synonymous mutation rates within the respective TCR regions, and in each case showing a high degree of amino acid conservation within the framework regions of the TCR.

Although the majority of CDR1 + 2 regions remained unchanged during evolution, a comparison of the number of mutations per site between species revealed a higher number of both synonymous and nonsynonymous mutations in the CDR 1 + 2 regions as compared to the framework region, again emphasizing the need to conserve those regions of the TCR necessary for proper protein folding. A previous study evaluating TCR evolution between primate species found similar results for the comparison of TCR sequences between Rhesus macaques and humans, with lower rates of synonymous and nonsynonymous mutations in framework compared with CDR regions of the TCR (21). In that study, a similar analysis comparing chimpanzee to human TCR sequences did not find significant differences in mutation rates of framework and CDR regions between species. However, the authors acknowledged that chimpanzee TCR sequence data were limited at the time of the study (20). Our access to a more complete set of chimpanzee TCR sequences allowed us to extend these results, and we provide evidence for purifying selection within the framework region of the TCR between human and chimpanzee lines.

One important difference between humans and chimpanzees involves the successful rearrangement of at least four TCRBV sequences in chimpanzees, which correspond to human pseudogenes. Two of the sequences, PtBV3-2 (PtBV9S2) and PtBV5-7 (Pt5S7), represent genes which belong to TCRBV families with two or more family members. The other two, PtBV21-1 (BV10S1) and PtBV23 (PtBV19S1), represent TCRBV families that are represented by only a single gene. Thus, no representatives of these two TCRBV families are expressed in humans but they are part of the TCRBV repertoire in chimpanzees.

This observation suggests that several TCRBV sequences have become nonfunctional over the five million years of evolution separating P. troglodytes from H. sapiens. Alternatively, an ancestral pseudogene could have transformed to a functional gene in chimpanzees. None of the mutations which are suspected to lead to functional impairment of the human BV21-1 (BV10S1) and BV3-2 (BV9S2) could be found in corresponding sequences derived from rhesus monkeys. This finding supports the conclusion that these pseudogenes were probably generated after the separation of the human and chimpanzee lineages.

Our data indicate that despite the high degree of similarity between human and chimpanzee TCRBV repertoires, differences due to the loss of genes over the course of evolution might influence the shape of the TCRBV repertoire in both species. Theoretically, these genes might be involved in the Ag-specific TCR repertoire and thus might have some functional relevance. Although the loss of TCRBV genes during evolution could lead to a narrowing of the overall TCRBV repertoire, the TCR repertoire may be highly redundant. When we screened HCV-specific T cell clones and lines for their TCRBV usage, we determined at least one of these functionally rearranged human pseudogenes, PtBV5-7 (BV5S7), contributes to the TCR repertoire directed against HCV in chimpanzees. It could be found in a cytotoxic T cell clone reactive against a MHC class I-restricted epitope and was present within the TCRBV repertoire of another CD8⁺ cell line. Another clonotype with a rearranged BV23 (BV19S1) gene was present in two HCV-specific T cell lines derived from a chimpanzee with resolved infection. These data demonstrate that these two variable chains contribute to the TCR repertoire against HCV infection and thus represent true "holes" in the human TCR V region repertoire. However, because there is no obvious impairment of human T cell responses as compared to chimpanzees the losses seem to be fully compensated by the remaining V regions.

In this study, we demonstrate a high level of conservancy between the TCRBV repertoires of chimpanzees and humans. Despite the high homology between TCRBV sequences, several functional chimpanzee TCR sequences that are part of the HCV-specific immune response have been lost over the course of evolution. In both humans and chimpanzees, strong HCV-specific T cell responses have been described, and the clearance rate of infection seems to be similar in both species. This is an indication that it is easy to compensate for the loss of single TCR genes and point to a high degree of redundancy of the TCRBV repertoire. Nevertheless, it has been hypothesized that the diversity of the TCRBV clonotypic responses to pathogens influences the outcome of infection (12, 13, 14). This relationship is especially important for complex T cell responses against highly mutable RNA viruses like HCV (9). The in-depth characterization of chimpanzee TCRBV genes in this study will facilitate the molecular analysis of the evolving immune response to pathogens in this important animal model.

	Acknowledgments

 
We thank George B. Cohen for helping in the design of primers used in this study and for providing technical assistance and advice.

	Footnotes

 
¹ This work was supported by Public Health Service Grants AI-RO1-A1-47367 and U19AI48231 (to C.M.W. and S.A.K.). S.A.K. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation. D.M.-O. is supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (Me 1891/1). N.H.S. was supported by postdoctoral fellowships from the Canadian Institute for Health Research and the American Liver Foundation.

² Address correspondence and reprint requests to Dr. Spyros A. Kalams, Vanderbilt University Medical Center, Medical Center North A4103, Nashville, TN 37232. E-mail address: spyros.a.kalams{at}Vanderbilt.edu

³ Abbreviations used in this paper: HCV, hepatitis C virus; CDR, complementarity determining region; TCRBV, TCR variable gene; KIR, killing-inhibitory receptor.

⁴ N. H. Shoukry, A. Grakoui, M. Houghton, D. Y. Chien, J. Ghrayeb, K. A. Reimann, and C. M. Walker. Memory CD8⁺ T cells are required for protection from persistent hepatitis C virus infection. Submitted for publication.

Received for publication November 13, 2002. Accepted for publication February 11, 2003.

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