• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of immunologyLink to Publisher's site
Immunology. Feb 2005; 114(2): 184–193.
PMCID: PMC1782068

Comparison of the expressed porcine Vβ and Jβ repertoire of thymocytes and peripheral T cells

Abstract

Transcripts of more than 300 unique T-cell receptor-β (TCR-β) V-D-J rearrangements recovered from porcine thymocytes and peripheral T cells were compared. We identified 19 groups (families) of porcine Vβ genes in seven supergroups and provisionally named 17 groups based on their sequence similarity with recognized human Vβ gene families. TRBV4S, 5S, 7S and 12S accounted for > 80% of all Vβ usage, and usage of these groups by thymocytes and peripheral T cells was highly correlated. No TRBV group was uniquely expressed in significant numbers in thymocytes, although small numbers of TRBV groups 2S, 9S and 15S were only recovered from T cells. Usage of Jβ segments from the 5′ D-J-C duplicon in thymocytes and peripheral T cells directly correlated with their 5′ position in the locus, and Jβ1·1, 1·2 and 1·3 accounted for ≥ 35% of all Jβ usage in both cell types. This contrasts with the usage of Jβ2 segments in that Jβ2·4, 2·5 and 2·7 accounted for ≈ 30% of Jβ usage by T cells and thymocytes. Jβ2·7 was threefold more frequent among T cells than thymocytes. The Vβ/Jβ combination was not random. Jβ1·1 and 1·2 were used in 29% of rearrangements with high frequency among the major Vβ groups. Combinations of TRBV4 and V12 with Jβ2·7 were only found in T cells and accounted for half of all Jβ2·7 usage. These studies show that unlike porcine heavy chain VH genes, the occurrence and relative usage of porcine TCR-Vβ groups resembles that of humans. Thus, highly related gene systems can individually diverge within a species.

Keywords: development, repertoire, swine, T cell, thymus

Introduction

αβ T cells are the most abundant peripheral T cells and are a major element of the adaptive immune system. Gene segments encoding both the γδ and αβ T-cell receptors (TCR) are highly conserved and arose early in vertebrate evolution.1 Like secreted immunoglobulins and the B-cell receptor (BCR), there are no genes encoding the polypeptides that comprise the TCR. Rather, gene segments are recombined under the influence of the same recombination activation genes (RAG) that are necessary for generating the BCR,2 although subsequent RAG-dependent receptor revision is controversial.3 Structurally, the TCR-β chain resembles both the VL and VH domains of immunoglobulins, and its framework regions are recognized by T-cell superantigens,4 much like B-cell superantigens that bind to the framework region of the BCR heavy chain.5 The latter observations, combined with its early expression as part of the pre-TCR6 and the use of diversity segments (Dβ), has earned TCR-β the vernacular title of the heavy chain of the αβ TCR.

The genomic organization of the Vβ locus and Vβ gene usage in mice and humans is well known.7,8 The Dβ-Jβ-Cβ region of the porcine Vβ locus is known from a BAC clone (GenBank AB079894), and some Vβ genes have been cloned from renal grafts.9 Nevertheless, the paucity of information on the Vβ genes and their usage in swine is surprising, given the importance of this species in diverse aspects of biomedical research. For example, fetal and neonatal piglets are models in developmental immunology.1012 Unlike mice and humans, the absence of in utero transmission of maternal antibodies, immune complexes and circulating antigen allow uncompromised studies on intrinsic development. The 114-day gestation provides a conveniently large window for studying fetal changes, and fetuses are larger than adult mice at gestation day 40 (DG40), so each is treated as a discrete statistical entity. The large fetuses also allow surgical manipulations to be carried out in utero.11 As offspring of this species are precosial, Caesarian-derived piglets can be reared in germfree isolators, allowing the influence of environmental microbes, dietary antigens and maternal factors on immunological development to be examined. Such studies have shown that colonization is needed to allow an immunoresponse to TD and TI-2 antigens,12,13 and certain Toll-like receptor (TLR) ligands can substitute for colonization.14 The accumulation of αβ T cells in the gut depends on colonization,15 and these are needed for the development of oral tolerance.16 Other biomedical swine models include those for rotavirus infections,1719 Escherichia coli O157,2022 influenza23 and Helicobacter pylori.24,25 Swine are also the choice for Neisseria/cilia interactions (M. Apicella, personal communication), for renal surgery26 and xenotransplantation.27

Selection as a model for medical research is not surprising because there is greater sequence similarity between human and swine genes/proteins than between those of humans and mice.2832 However, assumptions based on projections from data on human TCR genetics are unreliable because the constituency and usage of VH and VK genes in humans and swine differ markedly.30,3336 By contrast, studies on genomic organization, and similarities seen in Southern blots, suggest evolutionary conservation of TCR-β among species.1,37 However, neither Vβ usage nor Jβ usage is random in mice or humans,3842 immunoglobulin VH gene expression is non-random,4345 and VH gene usage may reflect the proximity of gene segments in the locus,4548 although not Vβ in mice.42 Biased Vβ usage is evident among thymocytes, suggesting that it is not the consequence of positive or negative thymic selection, but a result of recombination bias.4042 So far, T-cell studies in swine have been largely restricted to phenotypic analyses,49,50 so studies reported here and those on TCR-δ51,52 are designed to fill the molecular gap.

Here we characterize the expressed TCR-β repertoire in the peripheral T cells of conventional swine and compare this to the repertoire expressed by thymocytes. We identified 19 porcine Vβ gene groups (families) and named the 17 families that showed > 70% sequence similarity to human Vβ groups according to the human immunogenetic (IMGT) system.53 The same five Vβ families accounted for 85% usage in T cells and thymocytes. The > 300 complete TCR-β transcripts studied expressed all but one possible Jβ segment, and usage was highest (≈ 35%) among the 5′ Jβ1 segments. Heterogeneity in CDR3 was too great to allow identification of specific Dβ segments. This investigation reveals that the porcine Vβ repertoire is much more complex than the porcine VH repertoire and resembles the Vβ repertoire in humans.

Materials and methods

Source of tissue

Thymus, bone marrow and peripheral blood mononuclear cells (PBMC) were obtained from seven different animals: PBMCs from one newborn and from two young adults; mesenteric lymph nodes (MLN) from three neonatal animals; and thymus from one neonate and from one young animal. All animals were conventionally reared outbred swine and were healthy at the time of sample collection. The Vβ transcripts examined (see below) represent 30–50 from each of the seven different animals.

Preparation of RNA and cDNA for VβDβJβ transcripts

Tissues/cells for molecular biological studies of the TCR-β repertoire were placed in Tri-reagent (MRC, Cincinnati, OH) and stored at −70°. Total RNA was prepared, as previously described,54 and used as follows:

  1. To prepare poly A mRNA and subsequently a cDNA library (Stratagene, Palo Alto, CA).
  2. To recover expressed full-length TCR-β transcripts by reverse transcription–polymerase chain reaction (RT–PCR) by using 5′-RACE (Generacer; Invitrogen, Carlsbad, CA).
  3. To prepare cDNA using specific antisense Cβ primers for subsequent PCR cloning (see below).

We reasoned that the preparation and cloning of cDNA libraries would avoid the bias inherent in PCR-based cloning in which Vβ genes not recognized by degenerate leader primers would go undetected. We considered 5′-RACE to be the next best alternative. Once a reasonable sample of sequences, e.g. 100, was obtained by these methods, leader-specific primers could be used to rapidly expand the sample size by PCR cloning.

Cloning of Vβ genes

Rearranged Vβ gene segments, recovered as cDNA, were cloned into pCR4TOPO and plated on Luria–Bertani (LB) agar containing 100 µg/ml ampicillin (LB/AMP). Individual colonies were recovered, grown overnight and tested for an inserted Vβ gene by hybridization using a Cβ-specific probe and the same method used when cloning porcine VH genes.36 The strategy used was to recover a rearranged Vβ gene from the cDNA library by using 5′-RACE and appropriate primers (Table 2). Sequences of ≈ 100 rearrangements obtained in this manner were analysed to identify primer sites for PCR cloning. PCR cloning was performed by using leader primer sets that recognized certain Vβ gene supergroups (Table 1; Fig. 1). All clones containing inserts were sequenced and characterized as described below. Clones that did not contain a complete V-D-J rearrangement (≈ 50) were not included in the data presented.

Figure 1
Designation of porcine Vβ supergroups by similarity of leader (a) and variable gene (b) sequences. The 36 sequences selected for comparison represent all 19 apparent porcine Vβ gene groups identified in Fig. 2. The provisionally designated ...
Table 1
Oligonucleotide primers and probes used for cloning and identification of various Vβ gene groups
Table 2
Oligonucleotide primers used for cloning porcine Vβ genes using 5′ RACE

Sequence analysis

All cDNA products to be sequenced were cloned into pCR4TOPO and plated using TOP TEN cells in LB/AMP agar (see above). Relevant clones were selected on the basis of hybridization (see below), grown overnight in LB/AMP broth, tested for inserts of the correct size by restriction digestion and sequenced by using the Applied BioSystems (Foster City, CA) four-colour sequencer. Sequences were analysed by using Omiga (Accelys, Madison, WI) and the GCG system (Madison, WI). Alignment of clones recovered by this process was carried out by using GCG, and sequences were compared to those in the human Vβ IMGT database53 and to sequences recently reported by Baron et al.9 Sequences of Jβ were also compared to genomic sequences of Jβ in GenBank. The identification of framework regions (FR) conformational determining regions (CDR), and Dβ and Jβ regions/segments, was based on those described for human TCR-β.53

Nomenclature designation

The nomenclature adopted for the porcine Vβ and Jβ genes was that of Lefranc & Lefranc for human TCR genes. Naming of gene groups (families) was based on sequence similarity with consensus sequences of human Vβ groups, with thoughtful advice provided by Dr M. Lefranc (Table 3). An ‘S’ was inserted to denote provisional nomenclature.

Table 3
Sequence similarity of porcine Vβ gene groups (families) with those of humans

Previous sequence data

Vβ sequences recovered and partially published by Baron et al.9 were graciously provided by the authors. Unpublished sequences from Watanabe for Jβ, Dβ and Cβ were obtained from GenBank. Sequence for human Vβ genes, and those for mouse, were recovered from the IMGT Website and from GenBank.

Statistical analysis

Dr Kathryn Chaloner (Department of Biostatistics, The University of Iowa, IA) reviewed the data. Consultations suggested that all major differences in Vβ usage are apparent from inspection alone and that for less frequently used Vβ and Jβ segments, numbers were too low to consider testing for statistical significance. These considerations are stated whenever appropriate.

Results

Cloning of rearranged TCR-β genes

The cloning rationale described in the was as described below. Rearranged TCR-β transcripts were expected to be ≈ 1·3 kb. However, most clones obtained from the λZAP cDNA library were shorter and sequence analyses revealed them to be truncated in their 5′ regions. Therefore, we switched to the alternative use of 5′-RACE to recover a sufficient number of full-length cDNA transcripts that would offer the opportunity for identifying representatives of the major porcine Vβ families. Altogether, 122 full-length TCR-β transcripts were recovered from thymus and peripheral T cells by using 5′-RACE and library cloning. Sequence analysis of these representative transcripts revealed that the use of degenerate leader primers and antisense Cβ (Table 1) should allow all Vβ genes to be cloned by PCR. This was partially tested by comparing the relationship between the usage of different leader sequences and the usage of different Vβ gene sequences (Fig. 1). Information gained in this manner allowed degenerate leader primers and an antisense Cβ primer to be used to recover and sequence an additional 59 thymic and 153 peripheral TCR-β VDJ rearrangements. Altogether, > 400 clones were sequenced; data presented here are restricted to the analyses of 329 unique V-D-J rearrangements.

Porcine Vβ genes group to 19 families (groups)

All full-length sequences were compared in a dendrogram generated by using the GCG pileup program, and representatives of each tightly grouped set of sequences were then compared in the ‘old distances’ program, which allows percentage sequence similarity to be calculated but has a capacity of only 50 sequences. The results of analysing these representative sequences are given in Fig. 2. Figure 2 shows that 19 groups could be identified in > 300 full-length clones we recovered and among the 17 sequences kindly provided by Baron et al.9 Groups were designated on the generally accepted value of > 80% sequence similarity as a (family) group cut-off. The recovery of 19 Vβ group representatives among our clones is in very good agreement with the less extensive studies of Baron et al. We recovered only a single clone of nine groups (families), and Baron et al. recovered seven of these (Fig. 2).

Figure 2
Expressed porcine TRBV genes belong to 19 families. The old distances (GCG Package) was applied to representatives of the > 300 full-length unique porcine Vβ gene sequences that were first compared in the GCG dendrogram program. Two representative ...

Porcine Vβ gene exons fit to the same supergroups as their leader sequences

Figure 1(a) compares the leader sequences and Fig. 1(b) compares the Vβ sequences of all recovered TCR-β VDJ rearrangements. Comparing the sequences of various leader and Vβ exons allowed for the identification of distinct Vβ gene supergroups (I–VII). Supergroups established by grouping Vβ exons (Fig. 1b) compared favorably with those established by using leader sequences (Fig. 1a). A few exceptions were noted, namely that in Fig. 1(a), exon supergroups IIa and IIb are distinct but their leaders group together, and in Fig. 1(b), 4–1501 fits leader supergroup III but its Vβ exon does not group with any of the Vβ gene supergroup clusters. This outcome suggests that groups of expressed porcine TCR-Vβ gene families can be recovered by using primer sets comprising different leader-specific primers and an antisense Cβ primer (Table 1).

Apparent homologues of major human Vβ genes occur in swine

Table 3 presents a matrix that compares the consensus nucleic acid sequence of 19 porcine Vβ gene groups defined in Fig. 2 with those of major human Vβ gene families. pTRBVX, which contains VT100 (identified by Baron et al.9) and gT203 (identified by us), is not related to any human family, giving no better then 30–40% sequence similarity. Based on the results of Table 3, we have proposed a nomenclature for the porcine Vβ families (groups). Those sharing > 70% sequence homology with defined human Vβ groups were given the same group (family) name as in humans. However an ‘S’ was added, e.g. TRBV25S, to indicate that the porcine nomenclature is provisional. Thus, pTRBV2S may be derived from a common progenitor for both families. pTRBV2S had only 64% sequence homology to human TRBV2. Human Vβ groups with no ‘sequence homologues’ among the expressed porcine Vβ genes that we cloned, are not included in Table 3.

Four porcine Vβ groups are extensively used and some are multigenic families

It is clear, from inspection, that genes from pTRBV4S, 5S, 7S and 12S dominate the expressed repertoire in both T cells and thymocytes (Fig. 3a). Vβ genes from these four families, plus those from pTRBV21S, account for > 85% of the repertoire of the expressed repertoire. pTRBV7S alone accounted for 25% of the repertoire (Fig. 3a). In data to be published in greater detail elsewhere and in GenBank, the heavily used families also appear to be multigenic. pTRBV4S is represented by at least eight different genes, whereas heavily used families, such as pTRBV5S and pTRBV12S, appeared to be represented by alleles of a single V5S and V12S gene, respectively (J. E. Butler et al., unpublished).

Figure 3
(a) TRBV group usage in thymocytes and peripheral T cells in swine. The upper number on the x-axis designates the TRBV group, e.g. 4 = TRBV4S, while the lower number is the supergroup to which the group belongs (see Fig. 1). The number of sequences recovered ...

Usage of the major families is conserved in PBMC

Because the predominant usage of pTRBV-4S, -5S, -7S, -12S and -21S, shown in Fig. 3(a), could reflect a sampling error, we compared the usage of these five Vβ genes in 140 VDJ rearrangements from the PBMC of three unrelated animals: a newborn Landrace; an adult Landrace; and an adult Yorkshire. Figure 3(b) shows that differences in usage of the five genes or gene groups varied up to twofold among animals, but failed to show any age- or breed-related pattern. Overall, the combined usage varied from 76 to 94% of the total usage. Therefore, the predominant Vβ gene usage by PBMC cannot be explained by sampling bias.

Vβ genes of several families (groups) were only recovered from T cells

pTRBV2S, 9S and V15S genes were never recovered from thymocytes (Fig. 3a). This was also true for pTRBV3S, 10S, 19S, 29S and 30S. However the number of representatives obtained from these less frequently used genes was too small for using to test statistically. Regarding the opposite situation, no pTRBV family was exclusively recovered from thymocytes in substantial numbers.

Jβ segment usage differs between the 5′ Jβ-Cβ duplicon and the 3′ duplicon

The genomic organization of the human and mouse TCR-β locus is such that ≈ 65 Vβ genes belonging to 30 Vβ gene groups are located 5′ of a D-J-C duplicon at the 3′ end of the locus.53,55 Recent studies (GenBank, AB079894) indicate that the same organization exists in swine (Fig. 4b). Studies on immunoglobulin VH usage during development in mice,52 humans,51 rabbit56 and swine,54 indicate preference for the use of 3′ V genes and proximal D or J regions. Therefore, we addressed this question for TCR-β by comparing Jβ usage in 329 transcripts. Figure 4(a) shows a markedly skewed usage, in both thymocytes and T cells, of Jβ segments, which favours those at the 5′ end of the most 5′ D-J-C duplicon. Specifically, Jβ1·1, Jβ1·2 and Jβ1·3 account for ≈ 40% of the Jβ segments in thymocytes and for 30% of the Jβ usage by T cells. Usage of Jβ segments in the 5′ duplicon declined as their proximity to the upstream Vβ genes increased. J4β1·7 was never expressed. The pattern of Jβ usage within the 3′ duplicon differs from that in the 5′ duplicon. Specifically, there is equal or greater use of Jβ 2·4, 2·5 and 2·7 in T cells compared to Jβ2·1–2·4 (Fig. 4a). However, Jβ2·6 was less frequently used in both T cells and thymocytes than Jβ2·1–2·4. The combined usage of Jβ2·1, Jβ2·2, Jβ2·3 and Jβ2·6 was comparatively low (< 25%) and did not differ between T cells and thymocytes.

Figure 4
(a) TRBJ usage in thymocytes and in peripheral porcine T cells. The simplified Jβ-Cβ gene map shown in (b) for swine was constructed from sequence data in GenBank (accession number: ...

Jβ/Vβ rearrangements are non-random

The expression of certain Jβ/Vβ combinations appears to be non-random among more than 300 rearrangements examined (Fig. 5). For example, Jβ1·1 and Jβ1·2 were used in 94 rearrangements (29%) and with high frequency among the five Vβ genes that comprise > 85% of the repertoire (TRBV4, TRBV5, TRBV7, TRBV12 and TRBV21). A number of combinations were found only in T cells. These combinations include Jβ1·4/TRBV4, Jβ2·3/TRBV4, Jβ2·7/TRBV4, Jβ2·7/TRBV12 and Jβ1·1/TRBV21. Inspection alone suggests that at least three of these (highlighted by a shaded box with a black border in Fig. 5) cannot simply be explained by the higher proportion of T cells sampled. Noteworthy are the two combinations using Jβ2·7 because they account for half of all Jβ2·7 usage (44%). Although the numbers are too low to be statistically relevant, there are also examples of combinations found only in thymocytes. These include Jβ2·5 with TRBV20, TRBV21, TRBV25 and TRBV27.

Figure 5
The relationship between Vβ and Jβ usage in porcine T-cell-receptor-β (TCR-β) VDJ rearrangements in thymocytes and peripheral T cells. Shaded boxes with black borders indicate combinations that occurred in relatively high ...

Discussion

TCR-αβ T cells are numerically and perhaps the most important T cells in all species, even in ruminants and swine despite their high level of γδ T cells.57,58 Because the locus encoding the β chain of the heterodimeric αβ TCR is organized in segments encoding Vβ, Dβ and Jβ, a tendency persists to consider TCR-β as the TCR-αβ ‘heavy chain’, although some consider Cβ to be more closely related to Cλ, but Vα clusters with Vδ.55,59,60 As many features of BCR and TCR genetics differ among species,37,6062 the need exists to characterize each species. This is especially important for swine because of their use as models in basic research and infectious disease (see the Introduction), in xenotransplantation and as incubators for regeneration of human organs. Because the VH gene repertoire of swine and humans differs significantly,33,34 major differences in the Vβ repertoire might also be expected. In this report we identify the major groups (families) of expressed porcine Vβ genes and compare their usage, and the usage of Jβ segments, between thymocytes and peripheral T cells.

Data presented here show that 19 families (groups) of porcine Vβ genes could be identified by using the 80% cut-off criterion (Fig. 2), and that 17 of these share > 70% sequence similarity with defined families of human Vβ genes (Table 3). Therefore, and in accordance with the IMGT human nomenclature,53 these 17 families have been provisionally designated in the manner of their apparent human homologue, except that an ‘S’ has been added to indicate that the nomenclature is provisional (Table 3). Representatives of these families also cluster with representatives of TCR-β genes cloned from CD8 T cells infiltrating renal grafts by Baron et al.9 (Fig. 2). Of the two additional porcine Vβ gene groups, hTRBV2 shares 64% sequence similarity with the group provisionally named ‘pTRBV2S’, but the group labelled ‘pTRBVX’ has no better than 40% sequence similarity with the consensus sequence of any recognized human Vβ group. ‘Sequence homologues’ of nine additional recognized human Vβ families (TRBV 8, -13, -14, -16, -17, -18, -23, -24, -28) were not recovered from any of the porcine tissues examined. Vβ gene homologues have also been reported from other species, including sequence homologues in the chimpanzee, to those in humans63 and in goats.64 Thirteen families were reported for sheep, but their relationship to human families was not analysed.65 Designation of Vβ supergroups has practical and biological significance. First, the very similar grouping of leaders and Vβ segments suggests that these evolved and diversified together. Second, it guides the analysis of Vβ usage under various conditions by knowing which Vβ groups can be recovered by PCR using supergroup-specific leader primers (Tables 1 and and22).

The high sequence similarity of porcine Vβ gene families with human ‘sequence homologues’ suggests a common phylogenetic origin for these Vβ families. The high level of interspecies sequence similarity observed is consistent with the sequence homology of porcine and human major histocompatibility complex (MHC),28 various CH genes,29,31,32 and Vκ and Cκ.30 These results, and those of Uenishi et al.,66 suggest a closer phylogeny of swine to humans than humans to mice. However, other investigators reached a different conclusion,67,68 suggesting that mice and humans were more closely related than human and swine. This discrepancy may be explained if one accepts that all systems in a particular genetic progenitor (ancestor) do not evolve and diversify in parallel. Our studies support this position (see below).

It has been known, for some time, that the Vβ repertoire of thymocytes and peripheral T cells may differ because of negative69 and positive70 thymic selection. However, bias in Vβ usage appears to occur at the earliest stages of thymocyte development,4042 including the co-expression of certain Vβ and Jβ segments. For example, Vβ5 is preferentially expressed with J2·1, regardless of the stage of thymocyte development. Similar correlations were seen between the expression of Vβ12 and Jβ1·2 and 2·1, and between Vβ17 and Jβ1·5 and 2·1. Some evidence suggests that the commitment to a particular Vβ-Jβ usage is independent of thymic selection mechanisms.41 This observation is not universal, because Reinhardt & Melins71 showed apparent selection-induced repertoire skewing. Three examples of repertoire skewing were observed in our studies:

  1. Selective expression of certain Vβ gene groups by both thymocytes and T cells.
  2. Non-random usage of Jβ segments.
  3. Preferential Vβ and Jβ recombinations.

For example, just five groups (TRBV4S, V5S, V7S, V12S and V21S) out of 19 accounted for > 85% of the expressed Vβ repertoire of both thymocytes and T cells (Fig. 3a) and this appeared to be independent of age and breed (Fig. 3b). In non-stimulated human T cells, Vβ1, 3, 5, 12 and 13 are used with the highest frequency (Vβ1 is TRBV9 in the Lefranc nomenclature).72 We found no porcine sequence homologue of Vβ13, and Vβ3 was recovered in only one T-cell clone. However, TRBV5S and 12S accounted for > 30% of the expressed porcine repertoire. While we saw trends in skewed Vβ usage among cell types, the numbers were too low for statistical analysis. However, exclusive expression of TRBV9S and V15S was noted (13/0 and 6/0, respectively). We also observed repertoire skewing through the non-random usage of Jβ segments (Fig. 4a). For example, Jβ1·1, 1·2 and 1·3 together accounted for > 35% of all Jβ segments in both thymocytes and T cells. Non-random usage was also seen in the 3′ D-J-C duplicon, but in this case usage did not favour 5′ segments, and major differences were seen between T cells and thymocytes. For example, Jβ2·5 accounted for ≈ 14% of total Jβ usage in thymocytes, but < 10% in T cells. Jβ2·7 was recovered threefold more frequently from T cells than from thymocytes. The observation that biased Jβ usage carries over from thymocytes to T cells has been reported in mice and in humans.73,74 However, the degree of bias contrasts with studies in human fetuses in which 90% of the sequences used Jβ2 segments, not Jβ1 segments, as reported here.75 Finally, we observed skewing by the correlative usage of Jβ1·1, 1·2, 2·5 and 2·7, with the most highly expressed Vβ gene families in T cells. While Jβ1·2 and 2·5 were also preferentially used by the major Vβ families in thymocytes, Jβ2·7 was seldom used by thymocytes.

Our data indicate that, in contrast to the porcine heavy chain variable region repertoire, the αβ TCR ‘heavy chain variable region’, i.e. the Vβ repertoire of swine, is similar to that of humans in having multiple Vβ gene groups (families), 17 of which have > 70% sequence similarity to recognized human Vβ groups. If TRBV2 is included, 18 ‘sequence homologue’ families were recovered. Thus, related genetic systems do not follow a similar pattern of evolutionary divergence within the same species. This emphasizes the danger of extrapolations from overall genetic comparison67,68 without considering individual systems. As in other species, Vβ and Jβ usage is not random. While non-random over-representation of TRBV4S, 5S, 7S and 12S could reflect the number of genes present in these families, data to be published elsewhere indicate that TRBV4S contains at least eight genes, whereas pTRBV5S and pTRBV12S appear to be expressed as alleles of primarily one gene. The molecular and cellular basis for this non-random gene usage is a topic for future studies and could be especially relevant in understanding Vβ gene usage during pandemic diseases, such as porcine reproductive and respiratory syndrome virus,76 in this agriculturally important species, in swine models of human disease and in xenograft transplantation.

Acknowledgments

The authors are grateful for the assistance of Ms Marcia Reeve in the preparation of the typescript, to Dr Christian LeGuerin for kindly providing his library of porcine Vβ sequences, to Dr Marie Lefranc (Laboratoire d'ImmunoGenetique Moleculaire, Universite Montpellier) for her advice on naming the various porcine Vβ groups and families, to Dr Kathryn Chaloner for her advice concerning statistics, and to Dr Willi Born, National Jewish Medical Research Center, for his review of the manuscript. Research was funded by USDA-NRI grant no. 35204–10807.

References

1. Rast JP, Anderson MK, Strong SJ, Luer C, Litman RT, Litman GW. α, β, γ and δ T-cell antigen receptor genes arose early in vertebrate phylogeny. Immunity. 1997;6:1–12. 10.1016/S1074-7613(00)80237-X. [PubMed]
2. Yancopoulos GD, Blackwell TK, Suh H, Hood L, Alt FW. Introduced T cell receptor variable region gene segments recombine in pre-B cells. Evidence that B and T cells use a common recombinase. Cell. 1986;44:251–9. [PubMed]
3. Mostoslavsky R, Alt FW. Receptor revision in T cells: an open question. Trends Immunol. 2004;25:276–9. 10.1016/j.it.2004.04.001. [PubMed]
4. Choi Y, Herman A, DiGiusto D, Wade T, Marrack P, Kappler J. Residues of the variable region of the T cell receptor β chain that interact with S. aureus toxin superantigen. Nature. 1990;346:471–3. [PubMed]
5. Graille M, Stura EA, Cooper AL, Sutton BJ, Taussig MJ, Carbonnier JB, Silverman GJ. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci USA. 2000;97:5399–404. 10.1073/pnas.97.10.5399. [PMC free article] [PubMed]
6. von Boehmer H, Fehling HJ. Structure and function of the pre-T cell receptor. Annu Rev Immunol. 1997;15:433–52. 10.1146/annurev.immunol.15.1.433. [PubMed]
7. Rowen L, Koop BF, Hood L. The complete 695 kilobase DNA sequence of the human β T cell receptor locus. Science. 1996;272:1755–62. [PubMed]
8. Wilson RK, Lai E, Concannon P, Barth RK, Hood LE. Structure, organization and polymorphism of murine and human T cell receptor α and β chain gene families. Immunol Rev. 1988;101:149–72. [PubMed]
9. Baron C, Sachs DH, LeGuerin C. A particular TCRβ variable region used by T-cells infiltrating kidney transplants. J Immunol. 2001;166:2589–96. [PubMed]
10. Tlaskalova-Hogenova H, Sterzl J, Stephankova R, Balbac V, Veticka V, Rossmann P, Mandel L, Rejnek J. Development of immunological capacity under germfree and conventional conditions. Ann NY Acad Sci. 1983;409:96–13. [PubMed]
11. Butler JE, Klobasa F, Werhahn E, Cambier JC. Swine as a model for the study of maternal neonatal immunoregulation. In: Tumbleson ME, editor. Swine in Biomedical Research. Vol. 3. New York: Plenum Press; 1986. pp. 1883–9.
12. Butler JE, Sun J, Weber P, Francis D. Antibody repertoire development in fetal and neonatal piglets. III. Colonization of the gastrointestinal tracts results in preferential diversification of the pre-immune mucosal B-cell repertoire. Immunology (British) 2000;100:119–30. [PMC free article] [PubMed]
13. Butler JE, Weber P, Sinkora M, Baker D, Schoenherr A, Mayer B, Francis D. Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens. J Immunol. 2002;169:6822–30. [PubMed]
14. Butler JE, Francis D, Freeling J, Weber P, Nielsen KL, Krieg AM. TLR ligands act alone and synergistically to allow germfree piglets to respond to TI-2 and TD antigen. 7th IVIS 3; Quebec City. 2004. [PubMed]
15. Umesaki Y, Setoyama H, Matsumoto S, Okada Y. Expansion of α/β T cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germfree mice and its independence from thymus. Immunology. 1993;79:32–7. [PMC free article] [PubMed]
16. Sudo N, Sawamura SA, Tanaka K, Aiba Y, Kulo C, Koga Y. The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction. J Immunol. 1997;159:1739–45. [PubMed]
17. Iosef C, Chang KO, Azevedo MS, Saif LJ. Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pig model of human rotavirus disease. J Med Virol. 2002;68:119–28. 10.1002/jmv.10178. [PubMed]
18. Zijlstra RT, McCracken BA, Odle J, Donovan SM, Gelberg HB, Petschow BW, Zuckermann FA, Gaskins HR. Malnutrition modified pig small intestinal inflammatory response to rotavirus. J Nutr. 1999;129:838–43. [PubMed]
19. Yuan L, Saif LJ. Vet Immunol Immunopath. Vol. 87. 2002. Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model; pp. 147–60. [PubMed]
20. Francis DH, Collins JE, Duimstra JR. Infection of gnotobiotic pigs with Escherichia coli O157:H7 strain associated with an outbreak of hemorrhagic colitis. Infect Immun. 1986;51:953–6. [PMC free article] [PubMed]
21. Dykstra SA, Moxley RA, Janke BH, Nelson EA, Francis DH. Clinical signs and lesions in gnotobiotic pigs inoculated with Shiga-like toxin I from Escherichia coli. Vet Pathol. 1993;30:410–7. [PubMed]
22. Moxley RA, Francis DH. Overview of animal models. In: Kaper JB, O'Bried AD, editors. Escherichia coli O157:H7 and Other Shiga Toxin-Producing Ecoli Strains. Washington DC: ASM Press; 1998. pp. 249–60.
23. Van Reeth K, et al. Bronchoalveolar interferon-alpha, tumor necrosis factor-alpha, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? J Infect Dis. 1998;177:1076–9. [PubMed]
24. Krakowka S, Eaton KA. Helicobacter pylori-specific immunoglobulin synthesis in gnotobiotic piglets: evidence for the induction of mucosal immunity in the stomach. Vet Immunol Immunopathol. 2002;88:173–82. 10.1016/S0165-2427(02)00164-2. [PubMed]
25. Eaton KA, Cover TL, Tummuru MK, Blaser MJ, Krakowka S. Role of vacuolating cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1997;65:3462–4. [PMC free article] [PubMed]
26. Landman J, Olweny E, Sundoram CP, et al. Prospective comparison of the immunological and stress response following laproscopic and open surgery for localized renal cell carcinoma. J Urol. 2004;171:1456–60. 10.1097/01.ju.0000118649.56016.1c. [PubMed]
27. Simon AR, Warren AN, Sykes M. Efficacy of adhesive interactions in pig-to-human xenotransplantation. Immunol Today. 1999;20:323–30. 10.1016/S0167-5699(99)01485-1. [PubMed]
28. Singer DS, Camerini-Otro RD, Satz ML, Osborne B, Sachs D, Rudikoff S. Characterization of a porcine genomic clone encoding a major histocompatibility antigen: expression in mouse L cells. Proc Natl Acad Sci USA. 1982;79:1403–7. [PMC free article] [PubMed]
29. Brown WR, Butler JE. Characterization of Cα gene of swine. Mol Immunol. 1994;31:633–42. [PubMed]
30. Butler JE, Wertz N, Sun J, Wang H, Piumi F, Chardon P, Wells K. Antibody repertoire development in fetal and neonatal piglets. VII. Characterization of the pre-immune kappa light chain repertoire. J Immunol. 2004;173:6794–805. [PubMed]
31. Kacskovics I, Sun J, Butler JE. Five subclasses of swine IgG identified from the cDNA sequences of a single animal. J Immunol. 1994;153:3565–73. [PubMed]
32. Vernersson M, Pejler G, Kristersson T, Alving K, Hellman L. Cloning, structural analysis and expression of the pig IgE ε-chain. Immunogenetics. 1997;46:461–8. 10.1007/s002510050306. [PubMed]
33. Butler JE. Immunoglobulin gene organization and the mechanism of repertoire development. Scand J Immunol. 1997;45:455–62. [PubMed]
34. Sun J, Hayward C, Shinde R, Christenson R, Ford SP, Butler JE. Antibody repertoire development in fetal and neonatal piglets. I. Four VH genes account for 80% of VH usage during 84 days of fetal life. J Immunol. 1998;161:5070–8. [PubMed]
35. Butler JE. Disparate mechanisms drive antibody diversity among mammals: an important addition to immunology textbooks. Trends Immunol. 2003;5:1–18.
36. Sinkora M, Sun J, Sinkorova J, Christenson RK, Ford SP, Butler JE. Antibody repertoire development in fetal and neonatal piglets. VI. B cell lymphogenesis occurs in multiple sites with differences in the frequency of in-frame rearrangements. J Immunol. 2003;170:1781–8. [PubMed]
37. Charmley P, Keretan E, Snyder K, Clark EA, Concannon P. Relative size and evolution of the germline repertoire of T-cell receptor β-chain segments in nonhuman primates. Genomics. 1995;25:150–6. [PubMed]
38. Akolkar PN, Gulwani-Akolkar B, Pergolizzi R, Bigler RD, Silver J. Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes. J Immunol. 1993;150:2761–73. [PubMed]
39. Arase H, Arase N, Ogasawara K, Good RA, Onoe K. An NK1-1+ CD4+8 single positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor Vβ family. Proc Natl Acad Sci USA. 1992;89:6506–10. [PMC free article] [PubMed]
40. Ema H, Cumans A, Kouriksky P. TCR-β repertoire development in the mouse embryo. J Immunol. 1997;159:4227–32. [PubMed]
41. Nanki T, Kohsaka H, Miyasaka N. Development of human peripheral TCRβJ gene repertoire. J Immunol. 1998;161:228–33. [PubMed]
42. Wilson A, Marechal C, Robsin-MacDonald H. Biased Vβ usage in immature thymocytes is independent of DJβ proximity and pTα pairing. J Immunol. 2001;166:51–57. [PubMed]
43. Gu H, Tarlenton D, Müller W, Rajewsky K. Most peripheral B cells in mice are ligand selected. J Exp Med. 1991;173:1357–71. 10.1084/jem.173.6.1357. [PMC free article] [PubMed]
44. Kraj P, Rao SPAM, Glas AM, Hardy RR, Milner ECB, Silberstein LE. The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells. J Immunol. 1997;158:5824–32. [PubMed]
45. Schroeder HW, Jr, Mortari F, Shiokawa PM, Kirkham RM, Elgavish RA, Bertrand FE., III Developmental regulation of the human antibody repertoire. Ann NY Acad Sci. 1995;764:242–60. [PubMed]
46. Huetz F, Carlsson L, Tonberg U-C, Holmberg D. V-region directed selection in differentiating B lymphocytes. EMBO J. 1993;2:1819–26. [PMC free article] [PubMed]
47. Schroeder HW, Jr, Haillson JL, Perlmutter RM. Early restriction of the human antibody repertoire. Science. 1987;238:791–3. [PubMed]
48. Yancopoulos GD, Desiderio SV, Paskind M, Kearney JK, Baltimore JF, Alt FW. Preferential utilization of the most JH-proximal VH segment in pre-B cell lines. Nature. 1984;311:727–33. 10.1038/311727a0. [PubMed]
49. Sinkora M, Butler JE, Rehakova Z, Sinkora J. Development of γ/δ thymocytes during ontogony in pigs: a flow cytometry study. J Immunol. 2000;165:1832–9. [PubMed]
50. Sinkora M, Sinkora J, Rehakova Z, Splichal I, Yang H, Parkhouse ME. Prenatal ontogeny of lymphocyte subpopulations in pigs. Immunology. 1998;95:595–603. 10.1046/j.1365-2567.1998.00641.x. [PMC free article] [PubMed]
51. Holtmeier W, Käller J, Geisel W, Pabst R, Caspary WF, Rothkötter HJ. Development and compartmentalization of the porcine tcrδ repertoire at mucosal and extraintestinal sites. The pig as a model for analyzing the effects of age and microbial factors. J Immunol. 2002;169:1993–2002. [PubMed]
52. Holtmeier W, Geisel W, Bernert K, Butler JE, Sinkora M, Sinkora J, Caspary WF. Prenatal development of the porcine TCRδ repertoire: dominant expression of an invariant T cell receptor δ chain. Eur J Immunol. 2004;34:1941–9. 10.1002/eji.200425055. [PubMed]
53. Lefranc MP, Lefranc G. The T Cell Receptor Fact Book ImMuno GeneTic (IMGT) database. San Diego, CA: Academic Press; 2003.
54. Butler JE, Weber P, Sinkora M, Sun J, Ford SJ, Christenson R. Antibody repertoire development in fetal and neonatal piglets. II. Characterization of heavy chain CDR3 diversity in the developing fetus. J Immunol. 2000;165:6999–7011. [PubMed]
55. Glusman G, Rowen L, Lee I, et al. Comparative genomics of the human and mouse T cell receptor loci. Immunity. 2001;15:337–49. 10.1016/S1074-7613(01)00200-X. [PubMed]
56. Knight KL, Becker RS. Molecular basis of allelic inheritance of rabbit immunoglobulin VH allotypes. Implications for the generation of antibody diversity. Cell. 1990;60:963–70. [PubMed]
57. Binns RM, Duncan IA, Powis SJ, Hutchings A, Butcher GW. Subsets of null and gamma delta T-cell receptor+ T lymphocytes in the blood of young pigs identified by specific monoclonal antibodies. Immunology. 1992;77:219–27. [PMC free article] [PubMed]
58. Hein WR, Mackay CR. Prominence of γ/δ T cells in the ruminant immune system. Immunol Today. 1991;12:30–4. 10.1016/0167-5699(91)90109-7. [PubMed]
59. Marchalonis JJ, Schluter SF, Bernstein RM, Chen S, Edmundson AB. Phylogenic emergence and molecular evolution of the immunoglobulin family. Adv Immunol. 1995;70:417–506. [PubMed]
60. McCormick WT, Tjoelker LW, Thompson CB. Avian B cell development: generation of an immunoglobulin repertoire by gene conversion. Annu Rev Immunol. 1991;9:219–41. 10.1146/annurev.iy.09.040191.001251. [PubMed]
61. Flajnik M. Comparative analyses of immunoglobulin genes: surprises and portent. Nat Rev Immunol. 2002;2:688–98. 10.1038/nri889. [PubMed]
62. Marchalonis JJ, Schluter SF, Bernstein RM, Hohman VS. Antibodies of sharks: revolution and evolution. Immunol Rev. 1998;166:103–22. [PubMed]
63. Meyer-Olson D, Brady KW, Blackaud JT, et al. Analysis of the TCR beta variable gene repertoire in chimpanzees: identification of functional homologs to human pseudogenes. J Immunol. 2003;170:4161–9. [PubMed]
64. Obexer-Ruff G, Fluri A, Hein W, Lazary S, Peterhans E, Bertoni G. Caprine T-cell receptor variable beta-chain (TCRV beta) repertoire analysis and potential applications in cowdriosis immune response studies. Ann NY Acad Sci. 1998;29:321–6. [PubMed]
65. Halsey WA, Jr, Palmer BE, DeMartini JC, Howell MD. Analysis of sheep T-cell receptor beta-chain heterogeneity. Immunogenetics. 1999;49:206–14. 10.1007/s002510050481. [PubMed]
66. Uenishi H, Hiraiwa H, Yamamoto R, et al. Genomic structure around joining segments and constant regions of swine T-cell receptor α/δ (TRA/TRD) locus. Immunology. 2003;109:515–26. 10.1046/j.1365-2567.2003.01695.x. [PMC free article] [PubMed]
67. Madsen O, Scally M, Douady CJ, et al. Parallel adaptive radiations in two major clades of placental mammals. Nature. 2001;409:610–4. 10.1038/35054544. [PubMed]
68. Murphy WJ, Eizirik E, Johnson WE, Zhang YP, Ryder OA, O'Brien SJ. Molecular phylogenetics and the origins of placental mammals. Nature. 2001;409:614–8. 10.1038/35054550. [PubMed]
69. Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell. 1987;49:273–80. [PubMed]
70. MacDonald HR, Lees RK, Schneider R, Zinkernagel RM, Hengartner H. Positive selection of CD4+ thymocytes controlled by MHC class II gene products. Nature. 1988;336:471–3. 10.1038/336471a0. [PubMed]
71. Reinhardt C, Melms A. Skewed TCRV beta repertoire in human thymus persists after thymic immigration: influence of genomic imposition, thymic maturation and environmental challenge on human TCRV beta usage in vivo. Immunobiology. 1998;199:74–86. [PubMed]
72. Lang R, Pfeffer K, Wagner H, Heeg K. A rapid method for semi-quantitative analysis of the human Vβ-repertoire using TaqMan PCR. J Immunol Methods. 1997;203:181–92. 10.1016/S0022-1759(97)00028-8. [PubMed]
73. Candeias S, Waltzinger C, Benoist C, Mathis D. The V beta 17+ T cell receptor: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells. J Exp Med. 1991;174:989–1000. [PMC free article] [PubMed]
74. Jorres R, Meo T. Few V gene segments dominate the T cell receptor beta-chain repertoire of the human thymus. J Immunol. 1993;151:6110–22. [PubMed]
75. Raaphorst FM, Kaijzel EL, van Tol MJ, Vossen JM, van den Elsen PJ. Non-random employment of Vβ6 and Jβ gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR beta chain rearrangement. Int Immunol. 1994;6:1–9. [PubMed]
76. Lemke CD, Haynes JS, Spaete R, Adolphson D, Vorwald A, Lager K, Butler JE. Lymphoid hyperplasia resulting in immune dysregulation is caused by PRRSV infection in pigs. J Immunol. 2004;172:1916–25. [PubMed]

Articles from Immunology are provided here courtesy of British Society for Immunology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...