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The Journal of Immunology, 2008, 181, 2597 -2609
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Role of Production Frequency in the Sharing of Simian Immunodeficiency Virus-Specific CD8+ TCRs between Macaques1

Vanessa Venturi*, Hui Yee Chin*, David A. Price{dagger},{ddagger}, Daniel C. Douek{ddagger} and Miles P. Davenport2,*

* Complex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Kensington, Australia; {dagger} Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff, United Kingdom; and {ddagger} Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In some epitope-specific responses, T cells bearing identical TCRs occur in many MHC-matched individuals. The sharing of public TCRs is unexpected, given the enormous potential diversity of the TCR repertoire. We have previously studied the sharing of TCR β-chains in the CD8+ T cell responses to two influenza epitopes in mice. Analysis of these TCRβ repertoires suggests that, even with unbiased V(D)J recombination mechanisms, some TCRβs can be produced more frequently than others, by a process of convergent recombination. The TCRβ production frequency was shown to be a good predictor of the observed sharing of epitope-specific TCRβs between mice. However, this study was limited to immune responses in an inbred population. In this study, we investigated TCRβ sharing in CD8+ T cell responses specific for the immunodominant Mamu-A*01-restricted Tat-SL8/TL8 and Gag-CM9 epitopes of SIV in rhesus macaques. Multiple data sets were used, comprising a total of ~6000 TCRβs sampled from 20 macaques. We observed a spectrum in the number of macaques sharing epitope-specific TCRβs in this outbred population. This spectrum of TCRβ sharing was negatively correlated with the minimum number of nucleotide additions required to produce the sequences and strongly positively correlated with the number of observed nucleotide sequences encoding the amino acid sequences. We also found that TCRβ sharing was correlated with the number of times, and the variety of different ways, the sequences were produced in silico via random gene recombination. Thus, convergent recombination is a major determinant of the extent of TCRβ sharing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A diverse range of different TCRs is synthesized during the maturation of T cells in the thymus. This process involves the recombination of the germline V, D, and J gene segments and the addition of nucleotides at the V(D)J junction. The potential diversity of TCRs produced by V(D)J recombination is enormous (>1015 (1)). However, these TCRs must subsequently pass selection processes both in the thymus and in the periphery, and only a few percent of those TCRs produced survive to exist in the naive TCR repertoire of an individual. At any given time, the total number of T cells in an individual (~108 for mice (2) and ~1012 for humans (3)) is many orders of magnitude less than the number that can potentially be produced by V(D)J recombination. Moreover, an even smaller diversity of TCRs (~106 for mice (4) and 107 for humans (3)) is present in the peripheral TCR repertoire. Nonetheless, this diversity appears adequate for the recognition of the large variety of Ags encountered by an individual.

The seeming excess of potential thymically generated TCR diversity compared with required peripheral TCR diversity leads to the expectation of largely different TCR repertoires between individuals (5). However, the sharing of TCRs between individuals is not a rare occurrence. Studies (6, 7) have reported a substantial overlap in the naive TCR repertoire (~18–27% overlap between two mice (6)). There have also been many observations of identical TCRs occurring in the T cell responses to Ags and specific epitopes in multiple MHC-matched individuals (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51). Such T cell responses are often referred to as public T cell responses. In contrast, T cell responses involving little sharing of TCRs between individuals are often referred to as private T cell responses. However, with adequate sampling of the TCR repertoire and a sufficient number of individuals, a spectrum in the number of individuals sharing TCR sequences can be observed (48, 52).

Public T cell responses have been observed in many different species, including humans (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) (reviewed in Ref. 53), monkeys (38), rodents (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 54), and rainbow trout (51). The sharing of TCRs between individuals has also been observed in a variety of immune responses, including both acute (8, 12, 33, 45, 46, 48) and persistent infections (10, 14, 15, 19, 20, 21, 25, 26, 27, 29, 30, 32, 34, 35, 36, 37, 38), autoimmune diseases (9, 11, 13, 16, 17, 18, 22, 23, 24, 49, 50, 54), alloreactive states (28, 31, 42, 43), and tumor rejection (41). Moreover, studies have suggested that public TCRs may play specific roles in many immune responses. In the CD8+ T cell responses to various epitopes of CMV and EBV, public TCRs are particularly prevalent (10, 20, 21, 25, 26, 27, 29, 30, 35, 36) and may be associated with the characteristic focusing of CMV and EBV epitope-specific TCR repertoires over time (36). Public CD8+ T cell responses, which have been observed in both HIV (32, 34) and SIV (38), may also have implications for the escape of particular epitopes from immune recognition (38, 55). However, despite this large variety of observed public T cell responses, public TCRs remain an enigma because it is not yet well understood how an identical TCR arises in the peripheral TCR repertoires of so many different individuals.

We have previously studied the sharing of TCR β-chain (TCRβ) sequences for murine CD8+ T cell responses to two H-2Db-restricted epitopes of influenza A virus, nucleoprotein 366–374 and acid polymerase 224–233 (48). In this study, we showed that the relative efficiency with which TCRβ sequences can be produced via V(D)J recombination is a good predictor of the spectrum of sharing of TCRβ sequences between mice (48). Production efficiency was assessed by analyzing the number of different nucleotide sequences encoding the epitope-specific TCRβ amino acid sequences and germline encoding of the TCRβ nucleotide sequences. We also used computer simulations of a random gene recombination process and observed large differences in the production frequencies of different TCRβ nucleotide and amino acid sequences. The efficiency with which a particular nucleotide sequence is generated depends on the variety of recombination mechanisms (i.e., different contributions from the germline genes and nucleotide additions) and the frequency with which each of these recombination events occurs; the latter is largely dependent on the number of nucleotide additions required (47, 48). Furthermore, the frequency at which a TCR amino acid sequence is produced depends on the variety of nucleotide sequences by which it can be encoded and the frequency of production of each of these nucleotide sequences. The variety of nucleotide sequences encoding a TCR amino acid sequence largely depends on the codon degeneracy of the amino acids in the V(D)J junction. Convergent recombination was also evident at the level of the TCR repertoire for the H-2Db-restricted influenza A virus nucleoprotein 366–374 epitope-specific response, in which many different TCR amino acid sequences conformed to a CDR3β amino acid motif (48). The consensus amino acids around the V(D)J junction in this CDR3β motif had the potential to be produced frequently due to the large variety of different ways that they could be made from the germline genes. The facilitation of variable TCR production frequencies by convergent recombination has been observed in many studies, in which two common characteristics of public T cell responses recur, as follows: 1) public TCR amino acid sequences often require fewer nucleotide additions (10, 40, 47, 48), and 2) public TCRs are often encoded by many different nucleotide sequences both within and between individuals (21, 30, 32, 34, 35, 38, 45, 46, 48, 50, 56).

The observed relationship between TCR sharing and TCR production relies on TCRs being produced at variable frequencies in the thymus. However, because public TCRs are studied in the peripheral immune response to an antigenic epitope, this implies that the hierarchy of the production frequencies of different TCRs is somehow maintained during thymic selection and into the naive T cell repertoire. Importantly, this does not imply that public TCRs must be more frequently selected than other TCRs. A recent study suggests that thymic selection does not preferentially select public TCRβs (7). Thus, preservation of the hierarchy of production frequencies suggests that thymic selection and peripheral selection are relatively random with respect to production frequency. With regard to the immune response, this does not imply that all frequently produced epitope-specific TCRs will respond in every individual, or that the most frequently produced TCR will either be the most dominant or the most shared. There are also many other important factors that determine the clonotypic constitution of an immune response, such as the structural and biophysical properties of the interaction between the TCR and its cognate peptide-MHC complex (57, 58, 59, 60, 61, 62, 63, 64, 65, 66) (reviewed in Refs. 67 and 68), T cell competition for Ag (69), and stochastic events (70). However, across an epitope-specific TCR repertoire, there does appear to be some preservation of the hierarchy of TCR{alpha}- or TCRβ-chain frequencies from gene rearrangement in the thymus through the pairing of the TCR{alpha}- and TCRβ-chains, thymic selection, and peripheral events, which makes it more likely that the same frequently produced TCRs will respond to a particular antigenic epitope in many individuals.

In the present study, we investigate the sharing of TCRβ sequences in Mamu-A*01-restricted CD8+ T cell responses to the SIV CM9 (CTPYDINQM; Gag, residues 181–189) and SL8/TL8 (S/TTPESANL; Tat, residues 28–35) epitopes in rhesus macaques. It has been shown previously that many TCRβ amino acid sequences involved in the responses to the SL8/TL8 and CM9 epitopes are common between different macaques (38). Thus, the SL8/TL8- and CM9-specific CD8+ T cell responses provide an opportunity to study TCR sharing in immune responses to a persistent infection in an outbred population. Our previous study (48) of TCR sharing was restricted to T cell responses during an acute infection in inbred mice. It is therefore important to demonstrate that the factors that determine TCR sharing in T cell responses in an inbred population are also determinants in an outbred population. Although the rhesus macaques in this study are matched at one MHC class I allele, there are other MHC molecules that differ between individuals; these will differentially influence thymic and peripheral selection of the naive T cell repertoire as well as the nature of the immune response itself.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
TCR repertoire data

The TCRβ sequences analyzed in this study were obtained in previous published (38) and unpublished (by us) studies of the CD8+ T cell responses to the TL8 (TTPESANL; Tat, residues 28–35) and CM9 (CTPYDINQM; Gag, residues 181–189) epitopes of SIVmac251 and the SL8 (STPESANL; Tat, residues 28–35) and CM9 epitopes of SIVmac239 in Mamu-A*01+ rhesus macaques (Macaca mulatta). The rhesus macaques from which the TCR repertoire data were obtained originated from two different colonies. Twelve of the macaques were obtained from Covance Research Products (38), and the eight vaccinated macaques were from the Wisconsin National Primate Research Center (71). The experimental procedures used to obtain the TCRβ sequences are described in detail in the original publication (38). Briefly, Ag-specific CD8+ T cells were purified based on binding to peptide-MHC class I tetramers. Cell sorting was followed by unbiased amplification of all expressed TCRB gene products and extensive sampling of subcloned PCR products.

Macaque germline gene segments

The original studies (38), in which the SL8/TL8- and CM9-specific TCRβ sequences were obtained, used human germline gene sequences to identify the Vβ and Jβ for each sequence because the macaque germline genes were not then available. In this study, we used macaque germline Vβ, Dβ, and Jβ genes extracted from the recently published macaque genome (72) available from the National Center for Biotechnology Information Rhesus Macaque Genome Resources website (http://www.ncbi.nlm.nih.gov/ genome/guide/rhesus_macaque/). The macaque TCRβ genes were identified by comparison with homologous human TCRβ germline genes obtained from the annotated human genome available from the National Center for Biotechnology Information Human Resources website (http://www.ncbi.nlm.nih.gov/projects/genome/guide/human/). The ImMunoGeneTics (73) nomenclature for TCR germline genes is used throughout this study.

The germline genes used for the analysis are similar to the human TRBV27 (BV14 in Arden nomenclature), and TRBV6-1 (BV13 in Arden nomenclature), TRBD1, and TRBJ1-5 genes. Thus, these macaque genes are referred to by the ImMunoGeneTics gene labels assigned to these human genes. The macaque gene sequences for TRBV27, TRBV6-1, TRBD1, and TRBJ1-5 are provided in Table I.


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  		Table I. The macaque TCR germline genes used in the analysis of the SL8/TL8- and CM9-specific CD8+ TCRβ repertoires

 
Identification of the germline Vβ and Jβ genes for each TCRβ sequence

The germline Vβ and Jβ genes involved in the production of each TCRβ sequence were identified by determining the percentage match between the germline genes and the TCRβ sequence. Percentage matches were calculated over regions no less than 30 and 21 bp for the Vβ and Jβ genes, respectively. There was another macaque TRBV6 germline gene that was homologous to the macaque TRBV6-1 gene (93.4% of 287 bp) and completely identical within CDR3. Thus, for many sequences, it was difficult to distinguish which of these two genes was used by the TCRβ sequences. For the purpose of this study, we used only the TRBV6-1 gene. The inability to distinguish between usage of the TRBV6-1 and this other TRBV6 gene does not affect the analysis of the V(D)J recombination mechanisms due to their identity within the CDR3.

Analysis of the sharing of TCRβ sequences

A TCRβ sequence was considered shared if an identical CDR3β amino acid sequence, together with identical Vβ and Jβ usage, was observed in multiple macaques.

Alignment of the TCRβ sequences with the germline genes

Each TCRβ sequence was aligned with the germline Vβ, Dβ, and Jβ gene segments to determine the minimum number of nucleotides that could have been added during production. This process involved initially aligning the germline Vβ gene at the 5' end of the TCRβ sequence and then aligning the germline Jβ gene at the 3' end of the TCRβ sequence. Single base differences between the TCRβ sequences and the germline genes that were many nucleotides outside the V(D)J junction were not counted as random nucleotide additions, owing to the small probability of so many nucleotides being randomly added to produce the same sequence as the germline genes. The germline Dβ gene was then aligned to the remaining nucleotides in the interval between the identified Vβ and Jβ gene segments, with a match to a string of two or more nucleotides considered as originating from the germline Dβ gene segment. It was assumed that only one of the two Dβ genes, TRBD1, was involved in the gene recombination with the TRBJ1-5 gene. Any nucleotides that were not identified as being from the germline Vβ, Dβ, and Jβ gene segments were counted as nucleotide additions.

Simulation of V(D)J recombination

We simulated the production, via random V(D)J recombination mechanisms, of TCRβ sequences using the TRBV27 and TRBJ1-5 germline genes and the TRBV6-1 and TRBJ1-5 germline genes. The method used is similar to that described previously (48). These two portions of the TCRβ repertoire were simulated independently, because insufficient information about biases in the Vβ and Jβ pairing process is available to simulate reasonably this step of the V(D)J recombination process. It was assumed, as in the TCRβ alignments, that only the TRBD1 gene could be involved in a recombination process involving the TRBJ1-5 gene. The choice of simulation parameters was guided by the results of the alignments of the observed TCRβ sequences with the germline genes. However, the actual distributions of the number of nucleotides deleted/added could not be used because the alignment process is biased toward the TCRβ sequences being near-germline (i.e., estimating minimal number of nucleotide additions). In addition, the epitope-specific TCRβ repertoires may also reflect the effects of thymic selection, peripheral survival, and Ag selection. Thus, the numbers of nucleotide deletions/additions were all randomly determined, reflecting a completely unbiased recombination process. The simulation allowed for up to 12 random nucleotide deletions from both the 3' end of the germline Vβ gene segment and the 5' end of the germline Jβ gene segment. Nucleotides were randomly deleted from both the 5' and 3' ends of the germline Dβ gene segment, allowing for deletion of up to the full length of the Dβ gene segment. This was followed by the random addition, between the germline Vβ and Dβ gene segments and Dβ and Jβ gene segments, of up to 12 nt in total. The base of each nucleotide was randomly determined. Simulations of the generation of portions of the TCRβ repertoire were performed using Matlab 7.0.1 (The MathWorks).

Analysis of the relationship between in silico TCR production and in vivo TCR sharing

The relationship between in silico TCR production and the in vivo spectrum in the number of macaques sharing the TCRβ sequences was analyzed for observed TCRβ sequences. TCRβ sequences that could not be made by the simulation because of deviations (i.e., likely allelic differences) from the available germline genes (Table I) that were outside the V(D)J junction were not included in this analysis. The TCRβ repertoire data consisted of 31 of 1149 SL8/TL8-specific TRBV27/TRBJ1-5 TCRβ sequences, including both shared and unshared sequences, and 6 of 482 CM9-specific TRBV6-1/TRBJ1-5 TCRβ sequences with such deviations from the germline genes.

Statistical analysis

All correlations were performed using the nonparametric Spearman rank correlation and GraphPad Prism software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The SL8/TL8- and CM9-specific TCRβ repertoires

The TCRβ repertoire data available for this study consisted of 2949 SL8/TL8- and 3073 CM9-specific sequences obtained from 19 and 20 rhesus macaques, respectively. Our study focused on the 1149 TCRβ sequences using the TRBV27 and TRBJ1-5 genes and the 482 TCRβ sequences using the TRBV6-1 and TRBJ1-5 genes (Table II). The SL8/TL8-specific TCRβ repertoire preferentially uses the TRBV27 and TRBJ1-5 genes, and CDR3β amino acid sequences using these genes have been observed previously in multiple macaques (38). For the CM9-specific T cell response, Vβ gene usage is dominated by the TRBV6 group of genes (38, 74), but Jβ gene usage is more diverse than for the SL8/TL8-specific response (38). The TCRβ sequences using the TRBV6-1 and TRBJ1-5 genes were chosen for this study because they exhibited the greatest range in the number of macaques sharing TCRβ sequences.


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  		Table II. The characteristics of the SIV SL8/TL8- and CM9-specific CD8+ TCRβ repertoires

 
The pooled SL8/TL8-specific TCRβ repertoire from all 19 macaques consisted of 350 different CDR3β nucleotide sequences using the TRBV27 and TRBJ1-5 genes, which encoded 190 different TCRβ amino acid sequences (Table II). We observed the sharing of both TCRβ nucleotide sequences and amino acid sequences between macaques. Analysis of the sharing of TCRβ nucleotide sequences between macaques revealed that 7.4% of unique TCRβ nucleotide sequences were shared in the immune responses of more than one macaque. The most shared TCRβ nucleotide sequences were seen in five macaques. There was also substantial sharing of TCRβ amino acid sequences, with 20% of unique TCRβ amino acid sequences shared between two or more macaques. The most highly shared TCRβ amino acid sequence was present in 12 macaques. Importantly, rather than the commonly reported dichotomy of public and private TCRs, we observed a spectrum in the number of macaques sharing both TCRβ nucleotide and amino acid sequences (Fig. 1, A and C). Although the sharing of TCRβ nucleotide sequences was relatively low (7.4%), over half (54.9%) of the unique TCRβ nucleotide sequences encoded a shared TCRβ amino acid sequence (Fig. 1C).


Figure 1
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  		FIGURE 1. The spectrum in the number of macaques sharing TCRβ sequences in the SIV SL8/TL8- and CM9-specific CD8+ T cell responses. The distribution of unique TCRβ nucleotide (n.t.) sequences shared between a particular number of macaques for the SL8/TL8-specific TRBV27/TRBJ1-5 (A) and CM9-specific TRBV6-1/TRBJ1-5 (B) repertoires. The distribution of unique TCRβ nucleotide sequences encoding TCRβ amino acid (a.a.) sequences found in a particular number of macaques for the SL8/TL8-specific TRBV27/TRBJ1-5 (C) and CM9-specific TRBV6-1/TRBJ1-5 (D) repertoires.

 
Although there were fewer CM9-specific TCRβ sequences using the TRBV6-1 and TRBJ1-5 genes compared with the SL8/TL8-specific TRBV27/TRBJ1-5 TCRβ repertoire (482 vs 1149), a comparable proportion of this repertoire was shared between macaques (Table II). Of the 104 different CM9-specific CDR3β nucleotide sequences using the TRBV6-1 and TRBJ1-5 genes, 5.8% were found in more than one macaque. The most shared TCRβ nucleotide sequence was found in three macaques. Of the 62 unique TCRβ amino acid sequences, 21% were observed in more than one macaque. The most shared TCRβ amino acid sequence was present in seven macaques. Although the CM9-specific TCRβ sequences were not shared by as many macaques as the SL8/TL8-specific TCRβ sequences, a spectrum in the number of macaques sharing both TCRβ nucleotide and amino acid sequences was also observed (Fig. 1, B and D). Somewhat surprisingly, given that the CM9-specific TCRβ sequences were not found in as many macaques, almost half (47.1%) of the unique TCRβ nucleotide sequences using the TRBV6-1 and TRBJ1-5 genes encoded a TCRβ amino acid sequence that was shared (Fig. 1D).

Given that these macaques are an outbred population and that the total epitope-specific TCRβ repertoires were sampled (i.e., as opposed to sequencing epitope-specific TCRs with a specific Vβ, as in our previous study of TCRβ sharing in mice (48)), there was a high degree of TCRβ sharing in both the SL8/TL8- and CM9-specific responses. In the following sections, we investigate whether the observed spectrum of sharing among the SL8/TL8- and CM9-specific TCRβ sequences could be related to the frequency of production of these TCRβ sequences by germline gene recombination.

Shared TCRβ sequences involve fewer nucleotide additions

In an unbiased V(D)J recombination process, the more nucleotide additions required to produce a particular TCR nucleotide sequence, the less likely it is that the sequence will be made repeatedly by either the same recombination mechanism or a variety of recombination mechanisms. The reason for this is clear if one considers the probability of adding a nucleotide with the correct base during random nucleotide addition in the V(D)J junction. If 1 nt is added, there is only a one-fourth probability of adding in the required single nucleotide. If 2 nt are added, there is only a one-sixteenth probability of adding in the required 2 nt, and so on. Thus, one of the indicators that a TCR nucleotide sequence has the potential to be produced frequently by V(D)J recombination is that it requires fewer nucleotide additions (47, 48). Therefore, the potential for shared TCR sequences to be produced by gene recombination events involving fewer nucleotide additions would be supportive of a relationship between TCR production frequency and TCR sharing.

Although it is not possible to determine the actual recombination process involved in producing each observed epitope-specific TCRβ nucleotide sequence, we can estimate the minimum number of nucleotide additions between the aligned germline Vβ and Dβ, and Dβ and Jβ, gene segments. An example of the alignment of SL8/TL8-specific TCRβ sequences with the germline genes is given in Fig. 2. The maximum number of nucleotide additions required by both the SL8/TL8- and CM9-specific TCRβ nucleotide sequences was 11. Only two SL8/TL8-specific TCRβ nucleotide sequences could be made from the germline genes with no nucleotide additions. Both of these TCRβ nucleotide sequences encoded the same amino acid sequence, CASSLSRGSNQPQY, which was found in nine macaques. All CM9-specific TCRβ sequences required at least two nucleotide additions.


Figure 2
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  		FIGURE 2. An illustration of convergent recombination in the SIV SL8/TL8-specific CD8+ TCRβ repertoire. The SL8/TL8-specific CD8+ TCRβ repertoire using the TRBV27 and TRBJ1-5 genes consists of a majority of sequences conforming to the CASSXXRXSNQPQY amino acid motif and some nonconsensus amino acid sequences. A subset of each of the consensus and nonconsensus sequences is shown (A). At the level of the amino acid sequence, some amino acid sequences are found to be encoded by a variety of nucleotide sequences. This is demonstrated for the amino acid sequence CASSLSRGSNQPQY (yellow box), which was found to be encoded by 27 different nucleotide sequences (B). One possible alignment of the nucleotide sequences with the TRBV27 (blue), TRBD1 (red), and TRBJ1-5 (green) genes involving a minimal number of nucleotide additions (black) is shown for each nucleotide sequence. An additional level of convergent recombination is that some nucleotide sequences can be made many different ways and still require few nucleotide additions. Shown is an example of the multiple recombination events at the V(D)J junction, involving no more than two nucleotide additions, that can produce one of the nucleotide sequences (pink box) encoding CASSLSRGSNQPQY (C). The macaque germline genes are also provided (D). This illustration demonstrates that not only is CASSLSRGSNQPQY encoded by many nucleotide sequences, but also that some of these nucleotide sequences can be produced by a variety of V(D)J recombination mechanisms and that the production of many of these nucleotide sequences is made more favorable by the nucleotides at the junction ends of the germline genes. Moreover, some of these gene recombination events involve few nucleotide additions and are thus expected to recur frequently. Thus, the production via V(D)J recombination of the CASSLSRGSNQPQY amino acid sequence is enhanced by a convergence, across many different levels, toward encoding this particular TCRβ amino acid sequence.

 
We investigated the relationship between the germline encoding of TCRβ sequences and the sharing of TCRβ sequences between macaques. Significant negative correlations were found between the estimated minimum number of nucleotide additions and the number of macaques sharing the TCRβ amino acid sequences (SL8/TL8: r = –0.47, p < 0.0001; CM9: r = –0.45, p < 0.0001; Spearman) (Fig. 3, A and B). In addition to the sharing of TCRβ amino acid sequences, we also looked at the correlation between the minimum number of nucleotide additions and the extent of sharing of TCRβ nucleotide sequences. A similar relationship existed between the number of nucleotide additions and the sharing of TCRβ nucleotide sequences (SL8/TL8: r = –0.29, p < 0.0001; CM9: r = –0.33, p = 0.0006; Spearman). However, the correlation for the sharing of TCRβ nucleotide sequences was not as strong as for the sharing of TCRβ amino acid sequences.


Figure 3
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  		FIGURE 3. Analysis of the SL8/TL8-specific TRBV27/TRBJ1-5 and CM9-specific TRBV6-1/TRBJ1-5 TCRβ repertoires. The relationship between the estimated minimum number of nucleotide additions in SL8/TL8-specific (A) and CM9-specific (B) TCRβ sequences and the number of macaques in which an amino acid (a.a.) sequence was found responding to each epitope. The relationship between the number of unique nucleotide (n.t.) sequences encoding an amino acid sequence and the number of macaques in which a SL8/TL8-specific (C) and CM9-specific (D) TCRβ amino acid sequence was found. The opacity of the circles represents the density of points plotted. Also shown are the medians (horizontal lines) of the estimated minimum number of nucleotide additions or the number of unique nucleotide sequences encoding amino acid sequences. The correlation and significance values are based on the Spearman test.

 
In the CD8+ T cell responses to both the SL8/TL8 and CM9 epitopes, there was a significant trend for a TCRβ sequence found in more macaques to require fewer nucleotide additions in its production. This analysis suggests that, because TCR nucleotide sequences requiring fewer nucleotide additions have the potential to be produced more frequently by V(D)J recombination, the efficiency with which a TCR nucleotide sequence is produced plays a signficant role in TCR sharing.

Shared TCRβ amino acid sequences are encoded by multiple nucleotide sequences

There are several mechanisms that can lead to a TCR amino acid sequence being produced frequently by V(D)J recombination: it may be encoded by a frequently produced nucleotide sequence, it may be encoded by many different nucleotide sequences (for example, because the amino acids spanning the V(D)J junction are encoded by many codons), or a mixture of these two mechanisms may contribute to its efficient production. Thus, if shared TCR amino acid sequences are more frequently produced than unshared TCRs, then shared TCRs should be encoded by many more nucleotide sequences. The encoding of public TCRs by many different nucleotide sequences, both within an individual and across many individuals, has been observed in a number of previous studies (21, 30, 32, 34, 35, 38, 45, 46, 48, 50, 56). Moreover, it has been observed previously that shared SL8/TL8- and CM9-specific TCRβ amino acid sequences are often encoded by many different nucleotide sequences (38).

For the larger TCRβ repertoire data sets considered in this study, we determined the number of nucleotide sequences encoding each TCRβ amino acid sequence both within each macaque and across all macaques. We found that the maximum number of nucleotide sequences encoding a SL8/TL8-specific TCRβ amino acid sequence in a macaque was seven. The two SL8/TL8-specific TCRβ amino acid sequences, CASSLSRGSNQPQY and CASSLSRVSNQPQY, were both encoded by seven different nucleotide sequences in one macaque each. The SL8/TL8-specific TCRβ sequence, CASSLSRGSNQPQY, found in nine macaques, was encoded by a total of 27 different nucleotide sequences across all macaques (Fig. 2B). The SL8/TL8-specific TCRβ sequence, CASSLSRVSNQPQY, found in 10 macaques, was encoded by a total of 23 different nucleotide sequences across all macaques.

For the CM9-specific TCRβ repertoire, we found a maximum of five nucleotide sequences encoding a TCRβ amino acid sequence in a single macaque and a maximum of eight nucleotide sequences encoding a TCRβ amino acid sequence across the pooled repertoires of all macaques. The CM9-specific TCRβ amino acid sequence, CASSEAGNSNQPQY, found in five macaques, was encoded by five different nucleotide sequences within an individual macaque and by a total of eight different nucleotide sequences across the pooled repertoires of all macaques. An unshared TCRβ sequence, CASSGGGNSNQPQY, was also encoded by five different nucleotide sequences within an individual macaque. The sequence, CASSGQGNSNQPQY, found in three macaques, was another CM9-specific TCRβ amino acid sequence encoded by a total of eight different nucleotide sequences across all macaques.

Many of the highly shared SL8/TL8- and CM9-specific TCRβ sequences appeared to be encoded by a variety of different nucleotide sequences. We investigated whether the variety of different nucleotide sequences encoding the epitope-specific TCRβ amino acid sequences was related to the sharing of the TCRβ sequences. The number of macaques in which SL8/TL8- and CM9-specific TCRβ amino acid sequences were observed was found to be positively and significantly correlated with the number of nucleotide sequences encoding the amino acid sequences across all macaques (SL8/TL8: r = 0.91, p < 0.0001; CM9: r = 0.88, p < 0.0001; Spearman) (Fig. 3, C and D).

The observed relationship between the variety of nucleotide sequences encoding a TCRβ amino acid sequence and the number of macaques in which that TCRβ amino acid sequence was found is supportive of a trend for more frequently produced TCRs being more likely to be observed responding to a particular epitope in more individuals than other, less frequently produced, TCRs. Moreover, it suggests that the variety of different ways that a TCR sequence can be made, that is, convergent recombination, plays a role in some TCR amino acid sequences being more efficiently produced than others.

The sharing of TCRβ sequences conforming to the SL8/TL8-specific TCRβ amino acid motif

It has been observed previously that many of the public SL8/TL8-specific TCRβ sequences using the TRBJ1-5 gene conform to the amino acid motif CASSXXRXSNQPQY (38). The preferential usage of TCRβ amino acid sequences conforming to this motif, in which a variety of different Vβ genes is involved, suggests a strong structural preference for this CDR3β motif in this immune response. Analysis of the SL8/TL8-specific TCRβ repertoires using the TRBV27 and TRBJ1-5 genes revealed that 94.8% of unique nucleotide sequences encoding a shared TCRβ amino acid sequence also encoded the consensus CDR3β amino acid sequence. This remarkably high proportion suggests an association between the CASSXXRXSNQPQY amino acid motif and the sharing of these TCRβ sequences. With regard to the relationship between TCR production and TCR sharing, this association raises the question of whether the SL8/TL8-specific CDR3β motif has the potential to be made frequently by the V(D)J recombination process and thus be prevalent in the naive repertoires of many different macaques.

We investigated the potential for the conserved amino acids in the SL8/TL8-specific CASSXXRXSNQPQY motif to be germline encoded. We found that the CASS and SNQPQY portions of this motif can be fully encoded by the TRBV27 and TRBJ1-5 germline genes, respectively (Fig. 4A). Although the TRBV27 gene can also fully encode a leucine in the fourth CDR3 amino acid motif position (Fig. 4A), there was greater variability of amino acid usage at this position (Fig. 4B). Examination of the codon usage of the conserved arginine in the sixth amino acid motif position revealed that the arginine AGG codon was predominantly used (in 53.7% of unique TCRβ nucleotide sequences; Fig. 4C). This is the only arginine codon that can be fully encoded by the TRBD1 gene (Fig. 4D). We also found that the hierarchy of codon usage by the central arginine (Fig. 4C) appears to be largely determined by the variety of ways that the codons can be fully or partially contributed by the TRBD1 gene (Fig. 4, C and D). To assess whether the arginine AGG codon was predominantly germline encoded in the TCRβ sequences, we used our alignment procedure to estimate the length of the contributions from the TRBD1 gene. Of the TCRβ sequences using the arginine AGG codon in the sixth position of the CASSXXRXSNQPQY amino acid motif, we found that for 76.4% of sequences a string of at least 5 nt consisting of the AGG codon could be attributed to the TRBD1 gene (Fig. 4E). Thus, it is likely that the conserved arginine in many of the motif-related TCRβ sequences was fully or partially germline encoded by the TRBD1 gene.


Figure 4
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  		FIGURE 4. Analysis of the CASSXXRXSNQPQY amino acid motif prevalent in the SL8/TL8-specific TRBV27/TRBJ1-5 TCRβ repertoire. The germline encoding of the CASSXXRXSNQPQY CDR3β amino acid motif by the TRBV27 and TRBJ1-5 germline genes (A), the amino acid usage at the nonconsensus positions of the CDR3β amino acid motif (B), and analysis of the germline encoding by the TRBD1 gene of the conserved arginine in the motif (C–E). The potential encoding of the CASSXXRXSNQPQY amino acid motif by the TRBV27 (blue) and TRBJ1-5 (green) germline genes and the numbering of the CDR3β amino acid positions (A) are shown. The percentage of unique nucleotide sequences coding for a motif-related amino acid sequence that use each of the amino acids shown in the fourth, fifth, and seventh amino acid positions (B) is demonstrated. There is prevalent usage of leucine and serine in the fourth and fifth positions, respectively. This is largely due to germline encoding by the TRBV27 gene. The percentage of unique nucleotide sequences coding for a motif-related amino acid sequence that use each of the six arginine codons in the sixth amino acid position (C) is demonstrated. This hierarchy of codon usage is largely explained by the germline encoding of the various codons by the TRBD1 gene (D). The AGG codon (orange box) can be fully germline encoded, and there are also many ways that it can be partially encoded by the TRBD1 gene segment. The CGG codon (blue box) can also be partially encoded many different ways by the TRBD1 gene. There is a limited number of ways that the other arginine codons, AGA (green box), CGA (pink box), and CGC (purple box), can be partially germline encoded by the TRBD1 gene. Analysis of the potential germline encoding of the arginine AGG codon in the sixth amino acid position of the motif-related SL8/TL8-specific TRBV27/TRBJ1-5 TCRβ sequences (E) is shown. Shown are the percentages of unique nucleotide sequences coding for a motif-related amino acid sequence and using the arginine AGG codon for a specific number of nucleotides contributed by the TRBD1 gene. These percentages were determined using our alignment algorithm that attributes the largest and 5'-end-most match to the TRBD1 gene. Thus, in 4.9% of sequences, the AGG codon has not been attributed to the TRBD1 gene due to either a larger match to another portion of the TRBD1 gene or a match of 3 nt occurring before the central arginine position. The high percentage of sequences for which there are many nucleotides spanning the AGG codon that could be attributed to the TRBD1 gene demonstrates that it is likely that the AGG codon found at the central arginine position in the majority of motif-related TCRβ sequences is predominantly germline encoded by the TRBD1 gene.

 
This analysis suggests that the SL8/TL8-specific TCRβ motif, CASSXXRXSNQPQY, largely arises from germline-encoded conserved amino acids, with the variable amino acids occurring at the junctions between the truncated TRBV27 and TRBD1, and TRBD1 and TRBJ1-5, genes. Moreover, there is the potential for this pattern to arise frequently during gene recombination, which increases the likelihood that TCRβ sequences conforming to this motif would be present in the thymic and naive repertoires of many macaques.

Shared TCRβ sequences have the potential to be efficiently produced by V(D)J recombination

Our analysis of the SL8/TL8- and CM9-specific TCRβ repertoires demonstrates that the shared TCRβ amino acid sequences tend to be encoded by a greater variety of nucleotide sequences than the unshared TCRβs, and that these nucleotide sequences tend to require fewer nucleotide additions. This suggests that convergent recombination plays an important role in how efficiently a TCRβ sequence is produced, and that the frequency of production of a TCRβ sequence by V(D)J recombination is an influential factor in the observed spectrum of sharing of TCRβ sequences. However, it is difficult to assess the cumulative effect of convergent recombination at the different levels of the nucleotide sequence and amino acid sequence. Furthermore, alignment of the TCR sequences with the germline genes cannot determine the actual V(D)J recombination mechanism involved in generating a particular TCR nucleotide sequence and, hence, whether a prevalent TCR nucleotide sequence was produced by one frequently occurring V(D)J recombination event or a variety of V(D)J recombination mechanisms. We therefore used computer simulations of a random V(D)J recombination process. The number of times that the observed epitope-specific TCRβ sequences are generated in a simulation provides estimates of the relative production potential of these TCRβ sequences. These estimates enable us to address the following question: are the epitope-specific TCRβ sequences observed in the immune responses in more macaques produced more efficiently by unbiased V(D)J recombination?

The simulations involved the random removal of nucleotides from the 3' end of the Vβ, the 5' end of the Jβ, and both the 3' and 5' ends of the Dβ genes. This was followed by the random addition of nucleotides between the truncated Vβ and Dβ, and Dβ and Jβ, gene segments (more detail is provided in Materials and Methods). We generated 10 million in-frame sequences using each of the TRBV27 and TRBJ1-5, and TRBV6-1 and TRBJ1-5 gene combinations. From these simulated TCRβ repertoires, we were able to estimate how often each of the experimentally observed epitope-specific TCRβ sequences was produced via random recombination in silico, and compare this with the TCRβ sharing observed in vivo.

The number of macaques in which SL8/TL8- and CM9-specific TCRβ amino acid sequences were observed in vivo was found to be significantly correlated with the number of times the TCRβ amino acid sequences were produced in silico by the simulation of a random V(D)J recombination process (SL8/TL8: r = 0.32, p < 0.0001; CM9: r = 0.48, p = 0.0002; Spearman) (Fig. 5, A and B). Each point in Fig. 5, A and B, represents a TCRβ amino acid sequence observed responding to the SL8/TL8 and CM9 epitopes in vivo. The medians are shown to demonstrate that, although there is a lot of scatter in the data, the highly shared TCRβ sequences tend to be more frequently made in the simulations. Also, many of the TCRβ amino acid sequences that were found in one or two macaques were rarely, or never, generated in the simulations. For the SL8/TL8-specific response, the extent of sharing of TCRβ nucleotide sequences was correlated with the frequency with which they were generated in the simulation (r = 0.31, p < 0.0001; Spearman).


Figure 5
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  		FIGURE 5. Analysis of in silico TCRβ repertoires with respect to in vivo sharing of SL8/TL8-specific TRBV27/TRBJ1-5 and CM9-specific TRBV6-1/TRBJ1-5 TCRβ sequences. Shown are the following: the relationship between the number of macaques in which a TCRβ amino acid (a.a.) sequence was found in vivo and the number of times an amino acid sequence was generated in silico by the simulations (A and B); the number of different nucleotide (n.t.) sequences encoding the amino acid sequences in the simulation (C and D); and the number of different V(D)J recombination mechanisms in the simulation that produced the amino acid sequences (E and F) for the SL8/TL8-specific (A, C, and E) and CM9-specific (B, D, and F) responses. Each point on the graph represents an amino acid sequence that was observed experimentally in vivo in a particular number of macaques (horizontal axis). The opacity of the circles represents the density of points plotted. The horizontal lines represent the medians for the number of times (A and B), the number of nucleotide sequences encoding an amino acid sequence (C and D), or the number of different ways (E and F) that each amino acid sequence was generated by the simulation. The correlation and significance values are based on the Spearman test.

 
The analysis of the experimentally observed epitope-specific TCRβ repertoires revealed a significant correlation between the sharing of TCRβ amino acid sequences and the number of observed nucleotide sequences (as discussed in a previous section). This relationship was also reflected in the simulated TCRβ repertoires. For the observed epitope-specific TCRβ sequences that were generated in the simulation, there was a positive and significant correlation between the number of different nucleotide sequences encoding an amino acid sequence in the simulations and the number of macaques sharing the TCRβ amino acid sequence in vivo (SL8/TL8: r = 0.36, p < 0.0001; CM9: r = 0.39, p = 0.011; Spearman) (Fig. 5, C and D). We also assessed the cumulative effects, across the different levels of the nucleotide sequence and amino acid sequence, of convergent recombination on TCR production frequency. We tracked the variety of V(D)J recombination mechanisms producing each simulated TCRβ sequence. The number of different V(D)J recombination mechanisms producing the epitope-specific TCRβ amino acid sequences in silico was found to be significantly correlated with the observed sharing of the TCRβ amino acid sequences between macaques (SL8/TL8: r = 0.36, p < 0.0001; CM9: r = 0.40, p = 0.008; Spearman) (Fig. 5, E and F). Furthermore, it is clear from the vertical axes in Fig. 5 that many of the nucleotide sequences encoding the amino acid sequences were produced by many different V(D)J recombination mechanisms. For example, the SL8/TL8-specific TCRβ amino acid sequence, CASSLSRGSNQPQY, which was found in nine different macaques, was generated 1418 times in the simulations by 631 different V(D)J recombination mechanisms that resulted in only 163 different nucleotide sequences.

The simulation results demonstrate that an unbiased process of recombination of the germline Vβ, Dβ, and Jβ genes can give rise to a large range in the frequencies for different TCRβ sequences in the thymus. Furthermore, the variety of different ways that a TCRβ sequence can be made is an important determinant of the production potential of a TCRβ sequence. The correlations with the observed spectrum in the number of macaques sharing TCRβ sequences suggest that the potential efficiency with which TCRβ sequences can be produced plays a role in the sharing of the TCRβ sequences between macaques.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The sharing of identical epitope-specific TCRs between multiple MHC-matched individuals has been observed in a large variety of T cell responses (8, 10, 12, 13, 14, 15, 17, 19, 20, 21, 25, 26, 27, 29, 30, 32, 33, 34, 35, 36, 37, 38, 45, 46, 47, 48). The structure of the peptide-MHC complex and/or the TCR is often attributed as a determinant of both TCR sharing and TCR bias (i.e., clonal dominance), as discussed in recent reviews on this topic (67, 68). Public T cells have been observed responding to both prominent (hot and spicy) (58, 61, 63) and flat and featureless (vanilla) (57, 60, 62) peptide-MHC conformations, and some public TCRs have been observed undergoing conformational changes during the interaction with peptide-MHC (59, 66). However, Ag selection cannot explain how an identical TCR first came to be present in the naive repertoires of so many different individuals. Comparison of the TCR diversity estimates for the potential TCR repertoire (1) and the peripheral TCR repertoire of an individual at any given time (3, 4) suggests the presence of an identical TCR in the naive repertoires of so many different individuals to be unlikely. Our previous study (48) of TCRβ sharing between mice in CD8+ T cell responses to two influenza virus epitopes presented strong evidence for the frequency of TCR production playing a role in the sharing of TCRs between individuals and for variable TCR production frequencies being facilitated by a process of convergent recombination. In this study, we have performed similar analyses for the sharing of TCRβ sequences in CD8+ T cell responses to the SL8/TL8 and CM9 epitopes in SIV infection in rhesus macaques.

The SL8/TL8- and CM9-specific CD8+ T cell responses provide a challenging case to test the hypothesis that TCR sharing in an epitope-specific response is influenced by the contribution that TCR production frequency makes to the precursor frequencies of TCRs in the naive repertoires of each individual. These responses were studied in an outbred population of macaques. Thus, there are additional influences from different MHC class I molecules on the thymic, peripheral, and Ag selection of the epitope-specific TCR repertoire. Moreover, the total epitope-specific TCR repertoire (i.e., including TCRs using all Vβ genes) was sampled in each macaque. This provides less intensive sampling than a sample of the same size obtained from a TCR repertoire restricted to usage of a particular Vβ gene (as used in our previous studies of TCR sharing in mice (48)). The SL8/TL8-specific response also preferentially selects for TCRβ sequences that conform to a distinct CDR3β amino acid motif that is not strongly dependent on the usage of the Vβ gene. This is suggestive that the CDR3β sequence, or the TCR{alpha} sequence with which it pairs, provides a selective advantage in the CD8+ T cell response to the Mamu-A*01-restricted SL8/TL8 epitope. With regard to the TCR{alpha}, it was previously shown (38) that, although the SL8/TL8-specific response was dominated by the TRAV9-2 (AV22 in Arden nomenclature) gene, no strong consensus CDR3{alpha} sequence or shared CDR3{alpha} sequences were found.

Our analysis of the SL8/TL8-specific TCRβ repertoire using the TRBV27 and TRBJ1-5 genes and CM9-specific TCRβ repertoire using the TRBV6-1 and TRBJ1-5 genes revealed a high degree of sharing of TCRβ sequences between macaques. Greater than 5% of unique TCRβ nucleotide sequences and greater than 20% of unique TCRβ amino acid sequences were shared between macaques in both of the epitope-specific CD8+ T cell responses. However, compared with the SL8/TL8-specific TCRβ repertoire, the most highly shared CM9-specific TCRβ amino acid sequences were found in fewer macaques (7 vs 12 macaques). This could be partially due to the CM9-specific TCR repertoire being more clonotypically diverse (38). Moreover, we observed a spectrum in the number of macaques sharing epitope-specific TCRβ sequences. The SL8/TL8- and CM9-specific TCRβ amino acid sequences present in more macaques tended to be encoded by nucleotide sequences requiring fewer nucleotide additions and/or many different nucleotide sequences. Both of these characteristics are good indicators that the TCR sequences have the potential to be made efficiently. The relationship between the spectrum in the number of macaques sharing TCRβs and the production of the TCRβs via V(D)J recombination was further investigated using a simulation of this process. The simulation results demonstrated that the observed epitope-specific TCRβ sequences can be produced at variable frequencies of production, without the need for biases in the gene recombination process. Large differences in the production frequencies of different TCRβ sequences were facilitated by convergent recombination at the level of both the nucleotide sequence (i.e., the production of a nucleotide sequence by both recurring recombination events and many different V(D)J recombination events) and the amino acid sequence (i.e., many different nucleotide sequences encoding the same amino acid sequence). We found that the extent of sharing of SL8/TL8- and CM9-specific TCRβ sequences was significantly correlated with the in silico production frequency of these TCRβs via a completely random process of recombination of the germline genes.

A high proportion (~95%) of the unique nucleotide sequences encoding a shared SL8/TL8-specific TCRβ amino acid sequence was found to conform to the CASSXXRXSNQPQY amino acid motif. Closer examination of this motif revealed that the consensus amino acids can largely be germline encoded, with the variable amino acids spanning the junctions between the germline gene segments. Thus, this pattern of amino acid usage has the potential to arise frequently during gene recombination, because it is easier to obtain a string of nucleotides from a germline gene than for this string to be produced by random nucleotide additions. This characteristic was reflected in the codon usage of the central arginine in the CASSXXRXSNQPQY amino acid motif. The arginine codons that could be fully encoded (i.e., AGG), or partially encoded many different ways (i.e., CGG), by the TRBD1 gene were predominantly used in the motif-related SL8/TL8-specific TCRβ amino acid sequences. Thus, the prevalence of the motif-related SL8/TL8-specific TCRβ amino acid sequences between macaques is consistent with the potential of such sequences to be generated efficiently by the V(D)J recombination process.

The results of this study suggest that the frequency of production of TCRs by gene recombination is an important determinant of the sharing of TCRs between individuals in an immune response. This implies that the hierarchy of TCR production frequencies is maintained during thymic selection and into the naive repertoire, and subsequently reflected in the Ag-specific response. Moreover, we have demonstrated that the variable production rates contributing to this hierarchy of TCR frequencies are driven by a process of convergent recombination. Studies of the naive TCR repertoire, which require intensive sequencing of specific portions (i.e., defined by specific Vβ and Jβ genes) of the large and diverse pool of naive T cells, should enable further investigation of the contribution of TCR production frequencies to the naive precursor frequencies of individual TCRs. Understanding the relationship between TCR production frequency and TCR sharing has important implications for understanding the diversity and specificity of the T cell response both within individuals and across populations of individuals (55). In the presence of highly variable pathogens such as HIV and hepatitis C virus, the composition of the TCR repertoire may have important implications for immune escape and control of virus. The advent of high throughput TCR sequencing allows a more thorough investigation of TCR repertoire composition and evolution, although it is clear that much work is required to better understand TCR sharing and clonal dominance in immune responses.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the James S. McDonnell Foundation 21st Century Research Award/Studying Complex Systems, the Australian Research Council, and the National Institutes of Health. M.P.D. is a Sylvia and Charles Viertel Senior Medical Research Fellow, and D.A.P. is a Medical Research Council (United Kingdom) Senior Clinical Fellow. Back

2 Address correspondence and reprint requests to Dr. Miles P. Davenport, Complex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Kensington NSW 2052, Australia. E-mail address: m.davenport{at}unsw.edu.au Back

Received for publication April 23, 2008. Accepted for publication June 17, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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