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Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR ß-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets¹

Lars Helgeland^2,*, Finn-Eirik Johansen^*, Jon O. Utgaard^*, John T. Vaage and Per Brandtzaeg^*

^* Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, and Department of Anatomy, University of Oslo, Oslo, Norway

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR ß-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8⁺ subsets. Here, we have analyzed the TCR ß-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4⁺ as well as CD8⁺ subpopulations, and important differences were noted. CD8 and CD8ß single-positive (SP) IELs used most Vß genes, but relative Vß usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4⁺ IELs showed consistent skewing toward Vß17 and Vß19; these two genes accounted collectively for more than half the Vß repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4⁺ and CD8⁺ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping ß-chain repertoires. In conclusion, our results show that rat TCR-ß⁺ IELs of both the CD8⁺ and CD4⁺ subpopulations are oligoclonal. The limited Vß usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alarge population of lymphocytes is located between the villous epithelial cells in the small intestinal mucosa. These intraepithelial lymphocytes (IELs)³ consist of several different T cell subsets, some of which are found almost exclusively in this compartment 1, 2, 3, 4 . The presence of unique CD8 (TCR- or TCR-ß) single-positive (SP) and CD4/CD8 double-positive (DP) subpopulations has been proposed to reflect distinct origin and maturation pathways for IELs. Experimental studies in mice have suggested that such unique intraepithelial T cells as well as those expressing conventional CD8ß and CD4 SP phenotypes are extrathymically derived 2, 5 , but the thymic impact and the developmental relationship between the different subpopulations remain unresolved 1, 6 . In the rat we have recently shown that CD8ß and CD8 SP IELs populate the intestinal epithelium sequentially during the neonatal period in both the and ß T lineages, and that these CD8 populations dominate in young adult rats 7 . CD4 SP and CD4/CD8 DP IELs arrive later and accumulate with age, constituting substantial numbers in aged rats 8, 9, 10 . These IELs probably depend on the gut flora, as they are not found in germfree animals 10, 11 . The thymus appears to be critical for normal development and maturation of all CD3⁺ IEL subpopulations, because athymic nude rats contain few T cells with variably distorted phenotypes that are induced by the gut microflora only late in life 7 .

The role of intraepithelial T cells in mucosal immunity is poorly understood. In contrast to T cells in peripheral lymphoid organs, the functions of IELs appear to be conducted by a limited number of clones. Human TCR-ß⁺ IELs have been shown to express an oligoclonal receptor repertoire that differs among individuals 12, 13, 14 . Moreover, a murine study reported that the ß-chain repertoire differs even among genetically identical mice from the same litter. This finding was taken as evidence for a random and individually imprinted repertoire selection 15 . Most of these studies have focused on CD8⁺ IELs, and little is known about the CD4⁺ subsets. Indirect evidence in humans has suggested that CD4⁺ IELs are oligoclonal, as deduced from observations in unseparated colonic IELs that are relatively enriched in this subset 14, 16 .

Rat IELs have not been subjected to detailed repertoire analysis, but previously we investigated V region usage of TCR-ß IELs in AGUS rats with a small panel of Vß-specific mAbs 11 . Vß expression was found to be relatively unbiased and did not provide an indication of oligoclonality. However, with the same mAbs we recently obtained results compatible with oligoclonal expansions in PVG rats (unpublished observations), and the present report explores the ß-chain repertoire of sorted IEL subpopulations in this strain. Here, we show that although both the CD4⁺ and CD8⁺ subpopulations are oligoclonal, there are important differences between them. While CD8ß and CD8 SP IELs displayed variable ß-chain repertoires in different animals, both CD4 SP and CD4/CD8 DP IELs showed a consistent overexpression of Vß17 and Vß19. Furthermore, CD4 SP and CD4/CD8 DP IELs expressed overlapping ß-chain repertoires. These findings have significant implications for our understanding of the development and function of IELs.

	Materials and Methods

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Animals

Rats of the PVG strain were bred at the animal facility of the Department of Anatomy, University of Oslo (Oslo, Norway). They were reared under conventional conditions and routinely screened for common rat pathogens. Female rats were used at 5–6 mo of age.

Cell preparation and subset separation

IELs were isolated as previously described in detail 7, 11 . Briefly, the small intestine was flushed free of fecal content, inverted, and cut into 3- to 4-cm long pieces without disruption of the Peyer’s patches. After incubation twice for 15 min each time at 37°C in PBS with 0.1% EDTA, 0.3 mg/ml DTT, 5% FCS, and 1% antibiotics, cell suspensions were further enriched for IELs by centrifugation (30 min, 600 x g) on a one-step density gradient (Lymphoprep, Nycomed Pharma, Oslo, Norway). With this procedure, contamination with lamina propria lymphocytes that have a high content of B lymphocytes was estimated to be <2% 7, 11 . Also, the integrity of the remaining lamina propria was verified histologically (data not shown). In some experiments lamina propria lymphocytes (LPLs) were isolated. IELs were first removed, as described above, except for the excision of Peyer’s patches before EDTA/DTT incubation and the inclusion of an additional 15-min incubation at 37°C. LPLs were subsequently released by mechanical teasing as originally described by Lyscom and Brueton 17 , followed by Lymphoprep centrifugation. Lymph node lymphocytes (LNLs) were obtained from the mesenteric lymph nodes.

IEL subpopulations were prepared by FACSorting (FACS Vantage, Becton Dickinson, Mountain View, CA). For separation of CD8 SP IEL subsets, cells were stained with phycoerythrin-conjugated anti-CD8 (clone OX8), FITC-conjugated anti-CD8ß (341), and biotinylated anti-CD4 (OX35; all conjugates purchased from PharMingen, San Diego, CA). Binding of anti-CD4 conjugate was subsequently revealed with streptavidin-RPE-Cy5 (Dako, Glostrup, Denmark). Gates were set to exclude CD4⁺ IELs, and CD8⁺ IELs were separated into two fractions, CD8ß IELs (CD8⁺ß⁺) and CD8 IELs (CD8⁺ß^-). For separation of CD4⁺ IEL subsets, cells were stained with biotinylated anti-CD4 and FITC-conjugated anti-CD8 (Serotec, Oxford, U.K.) followed by streptavidin-phycoerythrin (Dako) and were subsequently sorted into CD4 SP (CD4⁺8^-) and CD4/CD8 DP (CD4⁺8⁺) IEL fractions. Control populations (CD8⁺ LNLs, CD4⁺ LNLs, or CD4⁺ LPLs) were purified in parallel by FACS. Sorted cells were checked for purity, which was routinely >98%. The numbers of recovered cells were: rat 1, 1.3 x 10⁶ CD8ß IELs and 0.9 x 10⁶ CD8 IELs; rat 2, 1.9 x 10⁶ CD8ß IELs and 1 x 10⁶ CD8 IELs; rat 3, 2 x 10⁶ CD8ß IELs and 1 x 10⁶ CD8 IELs; rat 4, 8 x 10⁵ CD8ß IELs and 2 x 10⁵ CD8 IELs; rat 5, 2.7 x 10⁵ CD4 SP IELs and 9.4 x 10⁵ CD4/CD8 DP IELs; rat 6, 2.9 x 10⁵ CD4 SP IELs and 6.7 x 10⁵ CD4/CD8 DP IELs; rat 7, 3.8 x 10⁵ CD4 SP IELs and 2.6 x 10⁶ CD4/CD8 DP IELs; rat 8, 1.9 x 10⁵ CD4 SP IELs and 5.9 x 10⁵ CD4/CD8 DP IELs; rat 9, 1.3 x 10⁵ CD4 SP IELs and 2 x 10⁵ CD4/CD8 DP IELs; rat 10, 1.7 x 10⁵ CD4 SP IELs and 2.6 x 10⁵ CD4/CD8 DP IELs; rat 11, 7 x 10⁴ CD4 SP IELs, 6.7 x 10⁵ CD4/CD8 DP IELs and 7 x 10⁴ CD4 SP LPLs; rat 12, 9.5 x 10⁴ CD4 SP IELs, 5.8 x 10⁵ CD4/CD8 DP IELs, and 1.1 x 10⁵ CD4 SP LPLs.

Preparation of RNA and cDNA

Total RNA was isolated from sorted cell populations by the guanidinium thiocyanate-phenol-chloroform method 18 in the presence of 100 µg of carrier transfer RNA. First-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase in enzyme buffer (10 U/µl; Promega, Madison, WI), dNTP (1 mM), rRNasin (1 U/µl; Promega), and oligo(deoxythymidine)₁₆ primer (1 mM) at 42°C for 60 min.

Analysis of TCR Vß usage by semiquantitative PCR

To determine Vß usage, cDNA was PCR amplified in 22 reactions corresponding to all known rat TCRBV subfamilies (Table I). Each 50-µl reaction contained approximately 1000 cell equivalents of cDNA, one of the Vß primers (0.6 µM) and a Cß primer (0.6 µM; 5'-TCTGCTTCTGAT GGCTCA-3') complementary to bases 42–59 of the first exon of rat TCRBC1 and TCRBC2 23 , in reaction buffer dNTP (0.2 mM), MgCl₂ (1.5 mM), Taq DNA polymerase (0.6 U), and enzyme buffer (Roche Molecular Systems, Branchburg, NJ). Aliquots of 15 µl were first heated to 95°C for 5 min and then subjected to 26, 30, or 34 cycles in a thermal cycler (Hybaid, Middlesex, U.K.); each cycle was 1 min at 95°C, 1.5 min at 55°C, and 2 min at 72°C. The PCR products, ranging from about 290–410 bp, were electrophoresed in 1.6% agarose gels and vacuum-blotted onto nylon membranes (Schleicher and Schuell, Dassel, Germany). Membranes were prehybridized in hybridization solution (5x SSPE, 2x Denhardt’s solution, 0.1% SDS, and 0.2 mg/ml salmon sperm DNA) in a hybridization oven for 3 h at 42°C. Hybridization was performed at 42°C overnight with an internal antisense Cß probe (5'-TGGGTGGAGTCACCGTTTTCAG-3') that had been end labeled with ³²P (1 x 10⁷ cpm/10 ml of hybridization solution), followed by washing in 1x SSPE/1% SDS for 30 min at room temperature and finally in 0.2x SSPE/1% SDS for 30 min at 35°C. Quantitation was performed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Initial analyses in LNLs showed that under these conditions, PCR amplification was in the linear phase between 26–32 cycles and reached a plateau at 35–37 cycles. Representation of individual Vß families was expressed as a percentage of the total signal obtained with all the Vß primers and was determined at the lowest number of cycles yielding a clear signal above background (usually 26 cycles).

TCRs were analyzed for distribution of V region lengths, resulting from the variable deletion and addition of nongermline-encoded nucleotides in the complementarity-determining region 3 (CDR3) during V-(D)-J joining. First, expressed Vß genes were PCR-amplified from 1000 cell equivalents of cDNA in 25-µl reactions for 34 cycles, using Vß- and Cß-specific primers under the conditions described above. PCR products were subsequently diluted 1/40 in 10-µl aliquots containing reaction buffer as described above and subjected to a 30-cycle run-off reaction with a ³²P end-labeled antisense Cß primer (0.13 µM; 5'-ATGGCTCAAACAAAGAGAC-3'). Each cycle was 1 min at 95°C, 1.5 min at 55°C, and 2 min at 72°C. Labeled primer extension products were heat denatured and separated on 5% denaturing polyacrylamide gels.

To visualize the distribution of CDR3 lengths in individual Vß-Jß combinations, antisense primers specific for rat TCRBJ gene segments were designed, avoiding the region upstream of the third residue 5' to the conserved phenylalanine (Table I). A similar approach has previously been described in the mouse 27 . Amplified PCR products were diluted 1/400 (to minimize intra-PCR recombinations) in 10-µl reactions containing 0.13 µM of a ³²P end-labeled antisense Jß primer and reaction buffer as described above and were subjected to 40 cycles of primer extension (each cycle was 1 min at 95°C, 1 min at 55°C, and 1.5 min at 72°C).

Cloning and sequencing of PCR products

To determine the DNA sequence of expressed Vß-chains, PCR products amplified with a set of Vß- and Cß-specific primers were either subcloned directly or after a nested PCR reaction where the Cß primer was substituted with a Jß-specific primer. PCR products were blunt ended with Pfu DNA polymerase, ligated into the SrfI site of pCR-Script (Stratagene, La Jolla, CA), and transformed into Epicurian coli cells according to the manufacturer’s instructions. Color-selected colonies were screened for insert of the correct size by PCR followed by agarose gel electrophoresis. Miniprep DNA was prepared by standard methods and was sequenced at Medigenomix (Martinsried, Germany) with the plasmid-specific oligonucleotide M13.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Variable skewing of the Vß repertoire in CD8 and CD8ß SP IELs

IELs from 6-mo-old PVG rats were isolated and analyzed for ß-chain repertoire, since at this age most IELs express the TCR-ß 28 . In accordance with previous data 7, 8, 9, 10, 28, 29 , 55–65% of IELs were CD8 SP, with the ratio between CD8ß⁺ and CD8⁺ cells being about 2:1. These two populations were sorted to approximately 99% purity while excluding CD4⁺ IELs. They were then subjected to Vß repertoire analysis by semiquantitative PCR. Fig. 1 shows Southern blots of RT-PCR-amplified Vß genes from rat 1. All Vß families were expressed by both CD8 SP IEL populations as well as by CD8⁺ LNLs, with the exception of Vß7. However, some Vß genes were expressed at relatively much higher and others at relatively much lower levels in IELs than in LNLs. Differences in Vß expression between CD8⁺ and CD8ß⁺ IEL were also noted, and these findings were substantiated by subsequent quantitation in four individual rats.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Vß gene expression in CD8 SP IELs and LNLs. Southern blots of RT-PCR-amplified Vß genes from CD8+ IELs (A), CD8ß+ IELs (B) and CD8+ LNLs (C) in rat 1. cDNA was amplified with Vß- and Cß-specific primers (26 cycles for A and C, 30 cycles for B), blotted onto nylon membranes, and hybridized with a 32P end-labeled internal Cß probe. Molecular size markers are indicated in base pairs.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Vß repertoire of CD8+ and CD8ß+ SP IELs and CD8+ LNLs from four individual rats (rats 1–4). Vß usage was quantified by PhosphorImager analysis after Southern blotting and hybridization with a 32P end-labeled Cß probe, as depicted in Fig. 1. Expression of each Vß gene is given as a percentage of the total signal produced by all the Vß primers as determined at a low cycle number yielding a clear signal above background (26 cycles, except for CD8ß+ IELs from rat 1 and CD8+ IELs from rat 3, which were determined at 30 cycles). In LNLs, Vß fractions were mostly in the 2–8% range with little individual variation, whereas in IELs Vß fractions were often in the 10–20% range with large variations between subsets and individual rats.

CD8 and CD8ß SP IELs are oligoclonal

Clonal status of CD8 and CD8ß SP IELs was investigated by analyses of CDR3 length distributions, which were visualized by subjecting RT-PCR-amplified TCR templates to run-off PCR reactions with a radiolabeled Cß primer, followed by separation on PAGE. Ten Vß subfamilies were selected for analysis. As expected, LNLs displayed multiple CDR3 lengths, the run-off reactions yielded between 6 and 10 bands (Fig. 3A). The spacing between the bands indicated that they were separated by three bases, corresponding to in-frame transcripts. Band intensities also were distributed evenly, typical of a polyclonal repertoire. This was not the case for IELs, where only one or a few prominent bands were visible for several Vß families, indicative of oligoclonal expansions. In a given IEL population, over-represented Vß genes yielded the most distinct oligoclonal patterns, as exemplified by Vß13 and Vß14 in rat 1. In the CD8⁺ IEL population, Vß13 accounted for 18% of the total Vß repertoire and comprised only one dominant band in the CDR3 length display. In the CD8ß⁺ IEL population, Vß13 constituted only 3% of the total repertoire but apparently contained TCRs with different CDR3 lengths (see Figs. 2 and 3A, respectively). The reverse pattern was seen for Vß14.

View larger version (80K): [in this window] [in a new window]   FIGURE 3. Oligoclonal ß-chain repertoires in CD8 SP IELs as judged by CDR3 length display. Vß-Cß PCR products from CD8+ LNLs (lanes a), CD8ß+ IELs (lanes b), and CD8+ IELs (lanes c) in rat 7 were subjected to run-off reactions with either a 32P end-labeled internal Cß primer (A), a Jß1.2 primer (B), or a Jß2.3 primer (C) before separation by PAGE. Run-off product sizes varied between Vß families, and they were therefore analyzed on two separate gels to achieve sufficient separation (Vßs 4, 13, 14, 15, and 19 were run for a longer time than Vßs 2, 8.2, 8.3, 16, and 20). Note the uneven distribution of bands in CD8ß+ and CD8+ IELs compared with CD8+ LNLs. In some lanes bands appear as doublets, presumably because of variable 3'-end adenosine extension by the Taq polymerase (30).

To confirm that dominant bands in the CDR3 length displays corresponded to mono- or oligoclonal populations, TCR transcripts from rat 1 were PCR amplified, cloned, and sequenced (Table II). In CD8⁺ IELs, all nine randomly isolated Vß15-Cß clones contained the same junctional sequence paired with Jß2.3. This indicated the presence of one dominant Vß15-expressing T cell clone and was consistent with the finding of only one band in the Vß15- Jß2.3 run-off reaction (Fig. 3C). Likewise, in CD8ß⁺ IELs 7/7 sequenced Vß4-Cß transcripts expressed Jß2.4 and showed identical junctional sequences (Table II). In CD8⁺ LNLs, on the other hand, the same primer pair yielded different junctional sequences in all isolates, as expected for a polyclonal population.

Detailed TCR repertoire analysis in CD4 SP and CD4/CD8DP IELs has not been reported. In accordance with previous rat data 7, 8, 9, 10, 28 , 20–30% of IELs expressed CD4, with approximately 25% being CD4 SP and 75% being CD4/CD8 DP. The latter subset expresses only the -chain, not the ß-chain, of CD8. The CD4 SP and CD4/CD8 DP IEL subsets were sorted to approximately 99% purity and subjected to Vß repertoire analysis by semiquantitative PCR. Fig. 4 shows representative Southern blots, in rat 5. Both the Vß17 and Vß19 families were markedly over-represented in CD4⁺ IELs. However, whereas CD4 SP IELs used all Vß genes, except for Vß7, this was not the case for CD4/CD8 DP IELs. In these cells, usage of some Vß genes was negligible.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Vß gene expression in CD4+ IELs and LNLs. Southern blots of PCR-amplified Vß genes from CD4/CD8 DP IELs (A), CD4 SP IELs (B), and CD4 SP LNLs (C) in rat 5. cDNA was amplified with Vß- and Cß-specific primers (30 cycles for A and C, 26 cycles for B), blotted onto nylon membranes, and hybridized with a 32P end-labeled internal Cß probe. Molecular size markers are indicated in base pairs. Note the increased expression of Vß17 and Vß19 in the CD4+ IEL subsets.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Vß repertoire of CD4 SP and CD4/CD8 DP IELs and of CD4+ LNLs in four individual rats (rats 5–8). Vß usage was quantitated by PhosphorImager analysis after Southern blotting and hybridization with a 32P end-labeled Cß probe, as depicted in Fig. 4. Expression of each Vß gene is given as a percentage of the total signal produced by all the Vß primers as determined at a low cycle number yielding a clear signal above background (26 cycles, except for CD4/CD8 DP IELs from rat 5 and CD4+ LNL from rats 5 and 6 determined at 30 cycles). Note the consistent skewing toward Vß17 and Vß19 in CD4 SP and CD4/CD8 DP IELs.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Vß repertoire of CD4 SP and CD4/CD8 DP IELs early after their appearance in the gut epithelium. Vß usage was quantified as described in Fig. 5 in two 3.5-mo-old rats (rats 9 and 10; determined at 26 PCR cycles). Note repertoire skewing toward Vß17 and Vß19.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Vß repertoire of CD4+ LPLs is not skewed toward Vß17 and Vß19. Vß usage was quantified as described in Fig. 5 in two adult rats (rats 11 and 12; determined at 26 PCR cycles). The Vß fractions ranged between 0.5–9%.

The results described above suggested that CD4⁺ IELs are oligoclonal, and this was confirmed by CDR3 length displays. In rat 5, analysis focused on the five Vß families 4, 15, 16, 17, and 19, which were well represented in the CD4/CD8 DP IEL subpopulation (see Fig. 5). As shown in Fig. 8, fewer bands were generally present for CD4/CD8 DP than for CD4 SP IELs in both the Cß and Jß run-off reactions; this further confirmed that the repertoire was more restricted in the former IEL subpopulation. Importantly, the data also suggested that CD4 SP and CD4/CD8 DP IELs expressed overlapping ß-chain repertoires, because the same dominant CDR3 lengths were usually present in both. This can be exemplified for the Vß17 and Vß19 families. Nested PCR amplifications showed that Vß17 was mainly coexpressed with Jß genes 1.4 and 2.3 and Vß19 was mainly coexpressed with Jß1.4 in CD4/CD8 DP IELs from rat 5 (data not shown). CDR3 length distribution with these two Jß genes showed bands of the same length in CD4 SP vs CD4/CD8 DP IELs (Fig. 8, B and C). Analogous results were obtained in three other rats (no. 6–8) for Vß17 and Vß19 in combination with Jß1.4 and Jß2.7 (Fig. 9).

View larger version (42K): [in this window] [in a new window]   FIGURE 8. Overlapping oligoclonal ß-chain repertoires in CD4 SP and CD4/CD8 DP IELs as judged by CDR3 length display. Vß-Cß products from CD4+ LNLs (lanes a), CD4 SP IELs (lanes b), and CD4/CD8 DP IELs (lanes c) in rat 5 were subjected to run-off reactions with a 32P end-labeled internal Cß primer (A), a Jß1.4 primer (B), or a Jß2.3 primer (C) before separation by PAGE. Note dominant bands of the same length in CD4 SP and CD4/CD8 DP IELs (lanes b and c).

View larger version (56K): [in this window] [in a new window]   FIGURE 9. Comparison of oligoclonal ß-chain repertoires in individual rats by CDR3 length display. Vß-Cß products from CD4+ LNLs (lanes a), CD4 SP IELs (lanes b), and CD4/CD8 DP IELs (lanes c) were subjected to run-off reactions with radiolabeled Cß primer (A), Jß1.4 primer (B), or Jß2.7 primer (C) before separation by PAGE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first detailed description of the TCR ß-chain repertoire of rat IELs. It has confirmed previous observations made in mouse and man that intestinal IELs are oligoclonal. In addition, it has revealed important differences between CD4⁺ and CD8⁺ IELs. The CD8 and CD8ß SP populations expressed a repertoire that was characterized by random oligoclonal expansions in individual rats. In contrast, in the same strain the repertoire of CD4 SP and CD4/CD8 DP cells was consistently skewed toward Vß17 and Vß19. This, together with the finding of overlapping ß-chain repertoires led us to conclude that the two CD4⁺ IEL populations are closely related.

Previous repertoire studies of IELs in mice and humans have mainly focused on the CD8⁺ subsets. The majority of human IELs are CD8ß⁺ and express TCR repertoires that differ between individuals 12, 13, 14 . However, little is known about the influence from genetic and environmental factors in shaping the oligoclonal repertoires in humans. This has been addressed in mice by the use of inbred animals kept under the same environmental conditions. Those studies analyzed separately the CD8⁺ and CD8ß⁺ IEL populations, but without excluding CD4⁺ cells. Nevertheless, they indicated that TCR usage of CD8 and CD8ß SP IELs was highly variable. No consistent patterns were observed by CDR3 length displays either between the two subsets or among individual animals 15, 34 . These data were taken as evidence that the gut epithelium is colonized with only a few randomly selected T cell clones. It was further believed that these clones expanded as a result of stimulation by luminal microbial Ags, as the appearance of TCR-ß⁺ IELs is dependent on the normal microflora. Also, the lack of correlation between the two CD8 SP subsets argued against the idea that CD8⁺ cells are a source of precursors for CD8ß⁺ IELs 15 . The present rat study has confirmed and extended the mouse data with respect to CD8⁺ IELs. The CD8 SP and CD8ß SP IEL subsets comprised clones of remarkably different sizes, which showed no correlation between the subsets or among individual animals.

Despite this result, some notable differences exist between the mouse and rat models. In the rat, stimulation with microbial Ags apparently is not required for development of TCR-ß⁺ IELs, because they are relatively abundant in germfree animals 11, 28 . Accordingly, colonization of the intestinal epithelium by TCR-ß⁺ cells takes place long before weaning and the establishment of a normal microflora, giving rise to a full complement of CD8⁺ IELs within the first week of life 7 . The vast majority of these neonatal T cells express both - and ß-chains of CD8, which raises the possibility that CD8⁺ SP IELs are derived from the CD8ß⁺ SP population 7 . Moreover, studies in chickens have suggested that neonatal IELs express a polyclonal, rather than oligoclonal, TCR repertoire 35 , and this may be the case in the rat as well. In keeping with this, reconstitution experiments performed in mice have shown that the first T cells to populate the gut epithelium express complex ß-chain repertoires 36 . Therefore, it would be interesting to know whether the oligoclonal TCR repertoires observed here in adult rats reflect clonal expansions over a diverse polyclonal background or the presence of only a limited number of clones in the epithelium. However, these alternatives are not easily distinguishable by PCR technology. At any rate, the nature of the Ags and/or restriction elements selecting the CD8 SP IELs subsets is difficult to deduce on the basis of repertoire analysis.

In this study CD4⁺ IELs were also subjected to separate TCR repertoire analysis. One striking finding was the predominance of Vß17 and Vß19 families in both CD4 SP and CD4/CD8 DP IELs. It is tempting to speculate that such Vß-specific expansions result from interactions with conserved microbial Ag, especially when considering that CD4⁺ IELs are induced by the microbial flora. Induction is especially pronounced for the CD4/CD8 DP population 10, 11, 37 . In line with this, CD4/CD8 DP IELs reportedly respond to heat shock proteins from both mycobacteria 38 and E. coli 39 , but only when these bacteria are present in the intestinal lumen. Bacterial superantigens might also conceivably stimulate IELs, e.g., when complexed with MHC class II molecules on epithelial cells 40 . However, as discussed by others 14 , superantigen stimulation alone would not expectedly give rise to oligoclonality, but only when combined with Ag-specific recognition events. It is also possible that Vß skewing reflects recognition of MHC class Ib molecules, e.g., CD1 or the thymus leukemia Ag that are expressed in the gut 41, 42, 43 . Some evidence to this end has been provided by employing TAP-deficient mice, which contained many CD4/CD8 DP IELs 44 . In any case, genetic background apparently influences Vß skewing in CD4⁺ IELs. Although preliminary experiments in another strain (AGUS) confirmed that the repertoire was skewed toward certain Vß genes, particularly in the CD4/CD8 DP subset, these animals did not show overexpression of Vß17 or Vß19 as did the PVG rats (unpublished data).

The origin and maturation pathways of IELs remain controversial. According to one theory, CD4/CD8 DP IELs arise from conventional thymus-derived CD4 SP T cells that migrate into the epithelium where they acquire CD8 expression. This possibility is supported by the observation that transfer of CD4 SP peripheral T cells into SCID mice reconstitutes CD4/CD8 DP IELs 45, 46 . Here, we have provided strong evidence for such a close relationship between the two CD4-expressing IEL subsets. Analysis of CDR3 length distribution as well as sequencing of TCR junctional regions demonstrated that CD4 SP and CD4/CD8 DP IELs express overlapping ß-chain repertoires. Our results also showed that the CD4/CD8 DP subset express a considerably more restricted repertoire than do CD4 SP IELs, consistent with continued intraepithelial expansion. On the contrary, our data fit poorly with the hypothesis that CD4 DP IELs are analogous to DP thymocytes that develop into mature CD4⁺ and CD8⁺ SP T cells 2 . Of all the IEL subpopulations analyzed, the CD4/CD8 DP subset expressed the most restricted ß-chain repertoire and was unrelated to the CD8 SP subsets. Therefore, it would be very difficult to view CD4/CD8 DP IELs as precursors for conventional positive and negative selection mechanisms.

	Acknowledgments

 
We thank Drs. E. Dissen and B. Rolstad for technical advice and support, and Ms. M. K. Johannesen for expert technical assistance.

	Footnotes

 
¹ This work was supported by the Norwegian Cancer Society, the Research Council of Norway, and the Anders Jahre’s Fund.

² Address correspondence and reprint requests to Dr. Lars Helgeland, Institute of Pathology, Rikshospitalet, N-0027 Oslo, Norway. E-mail address:

³ Abbreviations used in this paper: IELs, intraepithelial lymphocytes; SP, single positive; DP, double positive; LPLs, lamina propria lymphocytes; LNLs, lymph node lymphocytes; CDR, complementarity-determining region.

Received for publication July 2, 1998. Accepted for publication November 23, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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