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Development and Compartmentalization of the Porcine TCR Repertoire at Mucosal and Extraintestinal Sites: The Pig as a Model for Analyzing the Effects of Age and Microbial Factors¹

Wolfgang Holtmeier^2,*, Judith Käller^*, Wiebke Geisel^*, Reinhard Pabst, Wolfgang F. Caspary^* and Hermann J. Rothkötter

^* Medizinische Klinik II, Division of Gastroenterology, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany; and Center of Anatomy, Medical School of Hannover, Hannover, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells are an important component of the mucosal immune system. Previously, we have shown that the TCR repertoire in human intestine is polyclonal at birth and becomes increasingly restricted with age. In this study, we expand those studies to the pig which allows more extensive experiments including several organs. Tissues from different mucosal sites like the stomach, duodenum, ileum, Peyer’s patches, jejunum, and colon, and also extraintestinal sites like the lung, spleen, thymus and mesenteric lymph nodes, were obtained from conventionally reared pigs aged 2 wk to 5.5 years. In addition, tissues were also obtained from 10-wk-old specified pathogen- and germ-free pigs. TCRDV1-DV5 transcripts were amplified by RT-PCR after which complementarity-determining region 3 spectratyping was performed. Individual bands were excised from the gels and directly sequenced. The intestinal TCR repertoire showed increasing restriction with age and was highly oligoclonal in the adult 2- to 5.5-year-old pigs. In old pigs, we observed a striking compartmentalization. Different TCR repertoires were present between the lungs and the intestinal mucosa but also within different parts of the gastrointestinal tract. However, occasionally we observed identical TCR transcripts in the intestine and the lungs and shared clones could be detected also along the entire gastrointestinal tract. Thus, subsets of T cells are likely to transport immunological information between different compartments of the immune system. Furthermore, these data support the hypothesis that in each mucosal site, different Ags are responsible for selecting and maintaining the TCR over time.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of T cells is still unknown (1, 2). Recent insights into the structure of the TCR (3, 4) and the ligands recognized by human and murine T cells (2) strongly indicate that T cells have their unique functions. T cells are thought to form a first line of defense against the external environment because they are disproportionately enriched at mucosal sites (5). Furthermore, T cells share similarities with Igs and can recognize nonpeptide Ags in a non-MHC-restricted fashion (1, 6). It is now becoming clear that T cells can perform a vast number of immune effector functions and appear to play important roles in immune defense against pathogenic invaders in chronic inflammatory reactions, as well as in modulating systemic and organ-specific autoimmune diseases (5, 7). Furthermore, T cells might be critical in maintaining tissue homeostasis and epithelial integrity (8).

We have previously shown that the TCR repertoire in the intestine (9, 10, 11) and the skin (12) of adult humans is highly restricted and compartmentalized. In contrast, the intestinal TCR repertoire is polyclonal in the early period after birth (11), suggesting that intestinal T cells are shaped by selection over time. Moreover, an almost identical TCR repertoire was expressed at multiple different sites throughout the colon (11). It remains an open question which Ags drive the clonal expansions of T cells. Candidate Ags are foreign Ags like phosphoantigens or nonphosphorylated alkylamines which are found on microbes, or stress-induced self-Ags which are expressed on intestinal epithelial cells (2, 13, 14). For intestinal T cells, it is known that Ags from the normal flora are an important stimulus because their numbers do not increase in germ-free (GF)³ animals during the first 6 wk of life (15, 16).

To gain more insight into the biology of T cells, we chose the pig as an animal model for the following reasons: 1) the pig is one of the most important large animal models, and in contrast to rodents, repeated sampling of intestinal tissue and cannulation of the intestinal lymph duct is possible (17); 2) many physiological and pathophysiological data can be transferred to humans and research in other species is important to confirm murine data; 3) the main lymphoid cell populations of the pig are consistent with those of other vertebrates, especially those of humans (18); and 4) the immune system of the pig is of interest for veterinary as well as human biomedical research because the pig is increasingly recognized as a donor for xeno-transplantation.

Very little data are available about the TCR repertoire of the pig. This is largely due to the lack of mAbs which recognize V or V chains. In addition, there is still a lack of an Ab which recognizes all T cells (19, 20). Thus, currently the analysis is largely restricted to molecular biological methods. The constant region C chain was cloned several years ago (21) and only one paper defined TCRDV, DD, and DJ regions by sequencing cDNA and genomic DNA from the thymus of a 1-mo-old GF pig (22). No other organs or age groups have been studied until now. TCRDV regions were grouped into five families by the criteria of >75% nucleotide identity within the V regions. TCRDV1 consists of at least 15 members, DV3 and DV5 of two members, and DV2 and DV4 each appear to consist of a single gene. Furthermore, four different JD gene segments and three putative DD segments were described (22). Thus, the porcine TCR repertoire has the potential of an enormous recombinatorial diversity which is even greater than that described for humans and mice.

Our objective was to assess the diversity of the porcine TCR repertoire by complementarity-determining region 3 (CDR3) length spectratyping at different anatomical compartments to expand our previous human data and gain new insight into the biology of T cells. We were particularly interested in the distribution of intestinal T cells and therefore analyzed different anatomical sites along the intestinal tract and also extraintestinal sites. Pigs of different age groups were included to assess the postnatal development of the TCR. In addition, we analyzed the TCR repertoire in GF and specified pathogen-free (SPF) pigs to study the bias from the selective pressure provided by microorganisms. In this study, we report that the postnatal development of the porcine TCR repertoire strikingly resembles that of humans which makes the pig a useful animal model for elucidating mechanisms that control cell biology. Furthermore, porcine T cells showed a marked compartmentalization not only between different organs like the lung and the intestine, but also within the intestine, indicating that T cells at different locations recognize different Ags.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Samples

In the present study female pigs ranging in age from 2 wk to 5.5 years were used (see Table I). GF and SPF pigs were raised as previously described (23). Conventional pigs were kept under standard farming conditions. Samples from different anatomical compartments were taken from the animals at necropsy and snap frozen in liquid nitrogen and stored at -80°C until further use.

PCR amplification of TCR transcripts

Small tissue samples, 5–10 mm in size, were homogenized in 1 ml of TRIzol (Life Technologies, Karlsruhe, Germany). RNA was extracted under conditions recommended by the manufacturer and 1–2 µg total RNA were reverse-transcribed in a 20-µl reaction mix as described before (10, 11). TCRDV1-DV5 transcripts were amplified in 100 µl using 2–5 µl cDNA with 3.0 U Taq-polymerase (AmpliTaq Gold; PerkinElmer, Weiterstadt, Germany) and 25 pmol of V- and C-specific primer (see Table II). Amplification of TCR rearrangements consisted of 37 cycles of 40 s at 95°C, 50 s at 61°C, and 1 min at 72°C, followed by a final extension for 10 min at 72°C. Primers were designed according to the sequences given by Yang et al. (22) and Thome et al. (21). The TCRDV1 primer was designed to anneal in a conserved region which was shared by almost all known members of the DV1 family. The expected PCR product length was 200–250 bp.

For analysis of CDR3 lengths, 2–3 µl of each PCR mixture were added to formamide-containing loading buffer. PCR products were heat denatured for 3 min at 97°C. PCR products were then size-separated on a 6% denaturing polyacrylamide gel and visualized by silver staining (Silver Sequence DNA staining reagents) as recommended by the manufacturer (Promega, Mannheim, Germany). Bands were photographed by exposing polyacrylamide gels for 15–40 s to an automatic processor compatible film (Silver Sequence; Promega).

Direct sequencing of individual CDR3-length bands

For direct sequencing of TCR rearrangements, individual dominant bands were excised from the gels and incubated at room temperature in 50-µl sterile distilled H₂O, after which 5-µl aliquots were reamplified for 30 to 35 cycles using the same primers and PCR conditions described above. Double-stranded PCR products were directly sequenced using the ABI automatic sequencer 310 and the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase, FS (PerkinElmer), according to the conditions recommended by the manufacturer. For sequencing, the 3' primer TCRD seq. was taken (Table II). On several occasions, double bands, instead of a single band, were observed. These double bands are likely to be PCR artifacts (single adenine additions by Taq) or secondary to different migration properties of the two DNA strands (different conformation of the opposite strand) because sequence analysis of double bands gave only one sequence. Multiple additional dominant bands were selected where we could not obtain a readable sequence (data not shown). This was largely due to the fact that multiple different TCR transcripts of identical length were present within one band.

Sequence analysis and calculation of CDR3 lengths

Nucleotide sequences were analyzed using OMIGA software (Oxford Molecular, Cambridge, U.K.). The lengths of the CDR3 regions of translated TCR chains were calculated as previously described (10, 11, 24). This calculation is arbitrary because the exact borders of the CDR3 region are not defined and other groups use different calculations. In this study, CDR3 lengths were determined by the number of amino acids between the conserved cysteine C, which is encoded near the 3' end of TCRDV regions, and the conserved GXG triplet, which is encoded by all TCRDJ regions, and 8 aa were subtracted (24). The CDR3 region of the TCRDV4 sequence: CAVTGLSTFGGIQARNFDTDKLIFGKG, which is 27-aa long, has a CDR3 length of 19 aa (27 - 8 = 19).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The goal of this study was to perform a detailed analysis of the TCR repertoire in pigs of different ages (see Table I) and at multiple mucosal sites. Until now, only one paper analyzed the junctional diversity of TCR transcripts from the thymic RNA of a single, 1-mo-old GF pig which was reported to be highly diverse (22). No other organs or age groups were studied. In the current study, a total of 12 pigs (A–J) and 79 tissue samples were included. From each of the 79 samples, we analyzed the TCR repertoire of all known 5 DV families. To be capable to analyze 395 TCR repertoires (5 DV families x 79 tissue samples), we chose the method of CDR3 length analysis which visualizes the clonality of the TCR repertoire. Cloning and sequencing of such a large number of TCR repertoires is not a feasible approach because 16,000 sequences would have been needed to asses the diversity of the 395 TCR repertoires (40 sequences from each TCR repertoire). Using direct sequencing of selected CDR3 length bands enabled us to assess the TCR repertoire with a much smaller number of sequences (167 sequences were obtained).

The TCR repertoire is diverse in young pigs

In a first set of experiments, we analyzed the TCRDV1-DV5 repertoires of the lungs, the small and large intestine and the mesenteric lymph nodes in 2-wk-old conventional pigs (A and B). As shown in Fig. 1, the CDR3 length patterns were diverse for TCRDV1, DV3, DV4, and DV5 transcripts. This was independent of the tissue analyzed. A different picture was obvious when TCRDV2 transcripts were studied. CDR3 length spectratyping did not result in a normal length distribution and distinct bands were present. However, amplification of TCRDV2 transcripts by PCR gave a low yield, indicating that DV2 is rarely expressed. The same results and CDR3 profiles were obtained when a second primer pair and 10-fold more template cDNA was tried (data not shown).

View larger version (136K): [in this window] [in a new window]   FIGURE 1. CDR3 spectratyping of TCR transcripts from 2-wk-old pigs A and B. PCR amplified TCRDV1-DV5 transcripts from the left (L) and right (R) lung, the duodenum, the caecum, the rectum, and the mesenteric lymph nodes (Mes. LN) were size-separated on a denaturing gel. As shown, the CDR3 profiles of TCRDV1, DV3, DV4, and DV5 transcripts were close to a random length distribution, indicating a diverse TCR repertoire at this age. In contrast, TCRDV2 transcripts resulted in a restricted CDR3 length distribution.

The TCR repertoires become increasingly restricted in 10-wk-old pigs

We next analyzed the TCR repertoires of the small and large intestine, the mesenteric lymph nodes, the spleen and the thymus of 10-wk-old SPF pigs (Fig. 2a, C and D). The mucosal immune system of these pigs can be compared with that of conventional pigs except that their intestinal flora is free of pathogenic germs. Similar to the 2-wk-old conventional pigs, TCRDV1, DV4, and DV5 CDR3 patterns were diverse. However, some clonal expansions were visible on a polyclonal background within the TCRDV3 repertoires, and dominant TCRDV2 bands of identical length were found within the jejunum and ileal Peyer’s patches of pig C. Sequence analysis confirmed the same TCRDV2 transcript at both sites (sequence 8531; Fig. 3). The CDR3 profiles of the mesenteric lymph nodes, the spleen, and the thymus were highly polyclonal. Similar data were obtained from two additional conventional 10-wk-old pigs (G and H) where we analyzed small intestinal samples, mesenteric lymph nodes, and the spleen (data not shown).

View larger version (105K): [in this window] [in a new window]   FIGURE 2. CDR3 spectratyping of TCR transcripts from 10-wk-old SPF and GF pigs. a, From SPF pigs C and D, PCR-amplified TCRDV1-DV5 transcripts from the jejunum, Peyer’s patches of the ileum, caecum, mesenteric lymph nodes, spleen, and thymus were run concurrently on the same gel. As shown, CDR3 profiles of TCRDV1, DV4, and DV5 transcripts were highly polyclonal, whereas TCRDV2 and DV3 transcripts from the jejunum and the Peyer’s patches of the ileum exhibited multiple clonal expansions. Direct sequencing of the two dominant TCRDV2 bands with a CDR3 length of 17 (pig C) revealed one sequence (Fig. 3). b, From the GF pigs E and F, TCRDV1-DV5 transcripts were analyzed as described above. Similar to the SPF pigs C and D, clonal expansions were visible within the intestinal TCRDV2 and to a lesser degree within the DV3 repertoire. Dominant bands, as indicated by the CDR3 length numbers at the sides of the gels, were excised, reamplified, and directly sequenced (Fig. 3).

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Junctional sequences of TCR transcripts which were derived by direct sequencing from the dominant bands in Figs. 2 and 4. Putative TCRDD and DJ germline sequences are indicated at the top in boldface type (see text). GenBank accession numbers are AY112748–AY112755.

The TCR repertoire in 10-wk-old GF pigs is not significantly different from that in conventional or SPF pigs

To eliminate the bias from the selective pressure provided by microorganisms, we analyzed the TCR repertoires in the small and large intestine, the mesenteric lymph nodes, the spleen and the thymus of 10-wk-old GF pigs E and F (Fig. 2b). Similar to the 10-wk-old conventional and SPF pigs (Fig. 2a), clonal expansions were visible within the intestinal DV2 population and, to a lesser extent, in the DV3 population. The CDR3 profiles were highly diverse within the mesenteric lymph nodes, the spleen, and the thymus. Sequence analysis confirmed the same TCRDV2 transcript in the jejunum, ileal Peyer’s patches, and caecum of the GF pig E (sequence 8535; Fig. 3). In contrast, different TCRDV2 transcripts were found when dominant bands with a CDR3 length of 23 (sequence 8539, 8541) or 17 (sequence 8543, 8547) were sequenced from pig F.

The TCR repertoire is increasingly restricted at 28 wk of age and identical CDR3 patterns are visible throughout the small intestine

In the 28-wk-old minipig I (Fig. 4), multiple dominant bands were especially visible within the TCRDV3 repertoire of the small intestinal samples but also, to a lesser extent, within TCRDV2, DV4, and DV5 repertoires. Furthermore, the same dominant DV3 band was present in the jejunum, the jejunal Peyer’s patches, and the terminal ileum, indicating that the same T cell clones populate the entire small intestine. Sequence analysis of TCRDV3 confirmed identical transcripts when dominant bands of identical length were analyzed (sequence 8360; Fig. 3). Interestingly, the CDR3 profiles of the spleen, the mesenteric lymph nodes, and also the thymus remained highly polyclonal irrespective of the DV family analyzed. The highly polyclonal TCRDV1 CDR3 profile might be explained by the fact that DV1 consists of multiple members (>15) which were all amplified in one PCR. Thus, it is possible that clonal expansions within single DV1 family members are not visible because they are below the threshold of the method.

View larger version (76K): [in this window] [in a new window]   FIGURE 4. CDR3 spectratyping of TCR transcripts from 28-wk-old pig I; see also legends to Figs. 1 and 2. Multiple dominant bands were visible within the TCRDV2-DV5 repertoires of the small intestinal samples, whereas the CDR3 profiles of mesenteric lymph nodes, the spleen, and the thymus remained polyclonal. Direct sequencing of dominant bands, as indicated by the CDR3 length numbers to the left of the gels, revealed identical transcripts in the small intestinal samples (Fig. 3).

The TCR repertoire is highly oligoclonal and compartmentalized in 2- to 5-year-old pigs

We next analyzed the TCR repertoire in 2.5- and 5.2-year-old pigs J and K (Fig. 5). In contrast to younger pigs A–I, the CDR3 length profiles were highly restricted irrespective of the organ or DV family analyzed. In addition, we observed an almost fingerprint-like CDR3 profile which was typical for each organ and each individual pig. For example, the CDR3 profiles of TCR transcripts from the left and right lung were highly similar and sequence analysis confirmed identical nucleotide sequences (Fig. 6). Within the small intestine, we observed another CDR3 profile which was identical along the duodenum and jejunum, but distinct from that in the colon. This was independent of the DV region analyzed. From pig J we were able to study two additional samples from the terminal ileum. Whereas TCRDV1 and DV3 profiles of the jejunum and ileum were distinct from each other, there was no difference when DV4 and DV5 repertoires were studied. Thus, compartmentalization depends also on the DV family analyzed. We did not succeed in amplifying DV2 transcripts from the small intestinal samples in both pigs.

View larger version (112K): [in this window] [in a new window]   FIGURE 5. CDR3 spectratypes of TCRDV1-DV5 transcripts from a 2.5-year-old pig J (top) and a 5.2-year-old pig K (bottom); see also the legends for Figs. 1 and 2. The boxes with the patterns below the gels indicate which bands were sequenced and which contained identical dominant TCR transcripts. Blank boxes indicate no sequence. For example, the two dominant TCRDV1 bands with a CDR3 length of 17, which were found in the left and right lung of pig J, contained the same sequence (indicated as ; sequence 8155 is given in Fig. 6). The two bands of the same length, which were present in the duodenum and jejunum of the same pig, contained a different sequence (8159, indicated as ). This figure demonstrates that the TCR repertoire is oligoclonal and highly compartmentalized. However, occasionally dominant T cell clones can be found across different organs (e.g., pig J, DV3, CDR3 length 11).

View larger version (53K): [in this window] [in a new window]   FIGURE 6. TCR junctional sequences derived from dominant bands from pigs J and K (Fig. 5); see also the legend for Fig. 3. The origin of each sequence can be recognized by comparing the pattern in the third column with that in the boxes below the gels of Fig. 5. Sequences 8155 (DV1) and 8260 (DV4) were out of frame. DEL indicates the number of nucleotides which are deleted beyond the shown DJ germline sequence. GenBank accession numbers are AY112755–AY112792.

Sequence analysis of dominant bands with an identical CDR3 length revealed that the compartmentalization between different organs is not 100% (Figs. 5 and 6). For example, identical TCR transcripts could be isolated from the entire small and large intestine (Pig J, DV4 CDR3 length 12; pig K, DV3, CDR3 length 16 and 13), from the lungs and the colon (pig J, DV3, CDR3 length 11; pig K, DV2, CDR3 length 17), or from the lungs and the small intestine (pig K, DV4, CDR3 length 09). From pig K we also analyzed the mesenteric lymph nodes which exhibited an oligoclonal CDR3 profile which was distinct from those in the other organs. However, when we analyzed the dominant TCRDV1 band with a CDR3 length of 14, we found the same transcript in both the lungs and the mesenteric lymph node (sequence 8231). In contrast, the dominant TCRDV1 band with an identical CDR3 length from the colon contained a different sequence. Thus, dominant T cell clones can be present across different organs, although the CDR3 profiles indicate that each organ has its "private" TCR repertoire. These shared clones might represent recirculating T cells which are present in the lungs and the intestine.

The TCR repertoire is compartmentalized within the intestinal tract

From the 5.5-year-old pig L, we made a very detailed analysis from eleven different intestinal sites and also included samples from the stomach, the mesenteric lymph nodes, and the spleen (Figs. 7 and 8). Similar to pigs J and K, the TCR repertoire was oligoclonal in most organs and highly compartmentalized. However, we observed one TCRDV4 band (CDR3 length 19) which dominated all intestinal samples from the stomach to the sigmoid. In contrast, distinct CDR3 profiles were present in the different intestinal compartments when DV5 was analyzed. The TCRDV1 and DV3 profiles were more diverse, but restricted CDR3 profiles could be detected within each organ. Interestingly, the CDR3 profiles of the spleen were polyclonal (with the exception of DV2). Pig L was raised in a veterinary research institution and considered to be healthy. However, we cannot exclude a subclinical infection which might have caused the dominant TCRDV4 band. We do note that the TCR repertoires of DV1, DV3, DV4, and DV5 transcripts from the jejunal PP and jejunal mucosa were always identical. This was also the case in the younger pigs C, D, E, F, and J ( Figs. 2–4). Similar to pig J and K, we were unable to amplify TCRDV2 transcripts from the small intestinal samples of pig L.

View larger version (66K): [in this window] [in a new window]   FIGURE 7. CDR3 spectratyping of TCR transcripts from 5.5-year-old pig L. Similar to pigs J and K (Fig. 5), CDR3 profiles indicated that the TCR repertoire is oligoclonal and compartmentalized. However, this was also dependent on the DV region as shown by the dominant TCRDV4 band which was found across all intestinal samples. Sequences are given in Fig. 8.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. TCR junctional sequences derived from dominant bands from pig L (Fig. 7). TCRDV3 sequence 8314 was out of frame. GenBank accession numbers are AY112793–AY112805.

Molecular features of porcine TCR transcripts

Similar to humans, the junctional VDJ regions of porcine TCR transcripts were very complex with multiple N region additions, DD gene segment usage, and trimmed ends of the coding regions (Figs. 3, 6, and 8). Until now only the complete germline DNA sequences are known for TCRDJ1-DJ3. By comparing CDR3 sequences of TCR transcripts, Yang et al. (22) estimated the sequences of three TCRDD gene segments, the 5' end of the TCRDJ4 gene segment and the 3' ends of the TCRDV gene segments. Based on our larger number of TCR transcripts, we expanded the nucleotide sequences of the three TCRDD gene segments and the 5' end of the TCRDJ4 gene segment (see top of Fig. 3). TCRDJ1 was predominantly used, followed in frequency by DJ4. TCRDJ3 was used only twice and we never detected TCRDJ2. Translated amino acid sequences are shown in Fig. 9. The usage of single TCRDV1 family members was not random either. Because our primer allowed only the amplification of the very 3' end of TCRDV1 regions, it was not always possible to assign a single DV1 family member (22). V1.1 = sequence 8168 and 8159; V1.2 or 1.3 = sequence 8154, 8236, 8312; V1.8 or 1.10 = 8234, 8231, 8301, 8306; V1.11 or 1.12 = sequence 8162, 8230, 8237, 8307; V1.14 = sequence 8165 (Figs. 6 and 8).

View larger version (58K): [in this window] [in a new window]   FIGURE 9. Amino acid sequences from translated TCR transcripts which are shown in: a, Fig. 3; b, Fig. 6; and c, Fig. 8.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Similar to humans (11), we observed a diverse TCR repertoire in young pigs which became increasingly restricted and compartmentalized with age. The first clonal expansions of T cells were present within the intestinal TCRDV2 population at 2 and 10 wk (see below). At 6 mo, clonal expansions were also visible in other DV families (especially DV3). In contrast, the TCR repertoire of the extraintestinal sites like the mesenteric lymph nodes, the spleen, or the thymus remained highly polyclonal. Thus, the clonality of T cells is not a general feature of the entire organism and depends, at least during the first 6 mo after birth, on the expressed DV region. In the old pigs (up to 5.5 years), the TCR repertoire was highly oligoclonal irrespective of the expressed DV region and the same dominant clones were found along different sites of the small or large intestine. The homogenous, fingerprint-like CDR3 pattern of the TCR repertoire strongly suggests a model in which T cell clones, selected by ligands in the intestinal tract, undergo expansion and recirculation before lodging throughout the small or large intestine (11). A local expansion without recirculation would result in a more patchy distribution of clonally expanded T cells.

The role of TCRDV2-expressing T cells which exhibited a restricted repertoire in the early postnatal period remains unclear because mucosal TCRDV2 transcripts were barely detectable by PCR. Furthermore, we were unable to amplify any TCRDV2 transcripts from small intestinal samples of the old pigs. In contrast, TCRDV2 transcripts could be amplified without problems from the mesenteric lymph nodes, the spleen, and the thymus from the 10- and 28-wk-old pigs. These data indicate that DV2-expressing T cells are rare in the intestine whereas they are more abundant at extraintestinal sites. The restricted TCRDV2 repertoire can only be explained in part by PCR artifacts because identical, in-frame TCRDV2 transcripts were found at different colon sites. Thus, these dominant transcripts must have been derived from clonally expanded T cells.

In old pigs, T cells were highly compartmentalized and we observed distinct TCR repertoires not only between different organs like the lung and the intestine, but also within different parts of the gastrointestinal tract. Furthermore, this compartmentalization depended on the DV family analyzed. For example, within one pig distinct TCRDV5-expressing T cell clones were present in the stomach, the small intestine, and the colon whereas this was not the case for DV4 (Fig. 7). These data indicate that clonally expanded T cells are a heterogeneous population and that the expressed DV region might determine the role of that cell. It is imaginable that a T cell clone which is present along the entire gastrointestinal tract has another function than a T cell clone which is activated by an Ag which is only present in the stomach or colon.

The above findings again raise the important question about the recognized Ags. Recent data indicate that T cells are likely to be activated by stress-induced self Ags (2, 13, 14). It was hypothesized that the TCR might directly interact with stress Ags like MHC class I-related chain A/B which can be expressed in different organs. This could potentially lead to the oligoclonality and homogenous distribution of these cells. However, it was shown that the ligand for MHC class I-related chain A/B is not the TCR but the NK cell receptor NKG2D which is also expressed by most T cells (27, 28). The finding that the TCR might not directly recognize stress-induced molecules is in line with the observed compartmentalization of T cells. If all mucosal T cells would recognize the same stress-induced self Ags throughout the intestine, there would be no compartmentalization. It is rather likely that the TCR interacts with self Ags which are specific only for that site or with foreign Ags which are limited to that organ (for example, the microbial colonization of the colon is distinct from that in the small intestine).

To clarify the question of whether foreign Ags, like the intestinal flora, drive the clonal expansion of mucosal T cells, we analyzed GF pigs. During the first 6 wk of porcine life, there is a >10-fold increase of mucosal T lymphocytes (15, 16, 29). If Ags from the intestinal flora are responsible for the postnatal expansion of T cells, GF pigs would not be expected to express an oligoclonal TCR repertoire. However, as presented in this study, the diversity of the TCR repertoire from 10-wk-old GF pigs did not differ from that observed in age-matched, conventional, or SPF pigs and clonal expansions were likewise seen in the TCRDV2 population. The conclusions of this finding are in line with others which described that the localization of murine T cells to the intestinal epithelium is independent of the normal microbial colonization (30). However, the TCRDV2 repertoire was already restricted in 2-wk-old conventional pigs whereas the other DV families were polyclonal. Thus, older GF pigs (at least 20 wk old) must be studied to see whether the TCR repertoire of the other DV families becomes similarly restricted like that of conventional pigs. Unfortunately, this will be a difficult task because older GF pigs do not fit in the usual GF incubator.

A surprising finding was that bronchoalveolar T cells of the lower airways from the adult pigs J and K expressed a homogenous, oligoclonal TCR repertoire, and identical TCR transcripts were present in the left and right lung. Thus, these T cells might have similar functions to those of the intestine and are critical in elucidating their function as immunosurveillance cells of the lung (31). The immune response of the airways is particularly important because they are exposed to noxious stress, including infectious agents and environmental hazards. Murine studies suggested that T cells are critical for the negative regulation of airway responsiveness (32, 33). This was also hypothesized for intestinal T cells (7, 8). Thus, T cells might be responsible in maintaining homeostasis at different mucosal sites during immunoinflammatory challenge and postinflammatory repair (34). Our data of shared T cell clones between the lungs and the intestine support the notion that there is an ongoing recirculation and migration of T cells between the gut and the lung. This is in line with the observation that intestinal immunization with killed bacteria protects the lung against bacterial infection (35, 36, 37).

In summary, this study has deepened our understanding of which functions mucosal T cells might have. Furthermore, it will form the basis for future experiments studying the biology of T cells in a large animal. The similarity of the ontogeny and restriction of the porcine and human TCR repertoire indicate that the pig is a good animal model to study the function and homing mechanisms of T cells. It will be particularly interesting to see which Ags will be capable of changing the established oligoclonal TCR repertoire. Repeated sampling of intestinal biopsies or bronchoalveolar lavages (38) will be much more feasible in large pigs than in small mice. Furthermore, the migration of T cells can be studied after vaccination studies or infections of the gastrointestinal tract by cannulating the intestinal lymph and collecting the draining lymph at different time points (17).

	Acknowledgments

 
We thank Professor Waldmann, Klinik für kleine Klauentiere, Tierärztliche Hochschule Hannover and Dr. Klobasa, Bundesforschungsanstalt Neustadt-Mariensee for providing the organ samples of the adult pigs. We appreciate the technical help of Sunita Bahnik, Andrea Herden, Marita Peter, and Smjilka Schlecht.

	Footnotes

 
¹ W.H. was supported by Deutsche Forschungsgemeinschaft Grant Ho 1521/3-1 and by a grant from the Paul und Ursula Klein-Stiftung, Frankfurt am Main. H.-J.R. and R.P. were supported by Sonderforschungsbereich Grant SFB 280, Project C1.

² Address correspondence and reprint requests to Dr. Wolfgang Holtmeier, Medizinische Klinik II, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany, E-mail address: W.Holtmeier{at}em.uni-frankfurt.de

³ Abbreviations used in this paper: GF, germ-free; CDR3, complementarity-determining region 3; SPF, specified pathogen-free.

Received for publication March 20, 2002. Accepted for publication June 3, 2002.

	References

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