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The Journal of Immunology, 2006, 176: 3942-3949.
Copyright © 2006 by The American Association of Immunologists

Phenotypic and Functional Similarity of Gut Intraepithelial and Systemic T Cells in a Teleost Fish1

David Bernard*, Adrien Six{dagger}, Lionel Rigottier-Gois{ddagger}, Sébastien Messiaen*, Stefan Chilmonczyk*, Edwige Quillet§, Pierre Boudinot2,* and Abdenour Benmansour*

* Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France; {dagger} Unité d’Immunophysiopathologie Infectieuse, Institut Pasteur, Paris, France; {ddagger} Institut National de la Recherche Agronomique, Unité de Recherches Laitières et de Génétique Appliquée, Jouy-en-Josas, France; and § Institut National de la Recherche Agronomique, Laboratoire de Génétique des Poissons, Jouy-en-Josas, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3{epsilon}, TCR{zeta}, TCR{gamma}, and TCRbeta and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRbeta repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRbeta transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRbeta repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vbeta4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRbeta repertoire compared with the spleen and pronephros TCRbeta repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified {alpha}beta TCR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse {alpha}beta IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The gastrointestinal tract represents a major interface between the organism and the external environment. GALT includes specialized structures such as ileal and Peyer’s patches, and a great number of lymphocytes are distributed in the different layers of the mucosa. Both humans and mice possess a population of lymphocytes named intraepithelial lymphocytes (IELs)3 residing between intestinal epithelial cells (1). These lymphocytes have been extensively studied, because their permanent exposure to Ags implies that they should mediate tolerance and immunity to oral Ags and microflora (2, 3). Gut IELs consist of CD3+ T lymphocytes, which express either {gamma}{delta} or {alpha}beta TCR (4). This population contains predominantly CD8+CD4– lymphocytes but also significant numbers of CD8–CD4+ and CD8+CD4+, thereby differing from other peripheral T cell populations. The CD8 receptor expressed by mouse IELs consists of either heterodimeric {alpha}beta- or homodimeric {alpha}{alpha}-chains, and these markers identify functionally different T cell populations of distinct developmental pathways (4, 5). Regarding the diversity of the {alpha}beta TCR, the IELs express an oligoclonal repertoire in the adult individuals (6, 7, 8). Because the youngster repertoire seems to be polyclonal, selective mechanisms are probably responsible for the oligoclonality in human (9), rat (10), and chicken (11). A possible role of the gut microflora has been proposed (12, 13, 14) in these selective and developmental constraints.

The GALT of teleosts differs from the GALT of their mammalian counterparts in that it comprises only IELs scattered throughout the mucosa but no specialized structures similar to ileal or Peyer’s patches. Cells with lymphoid morphology residing between gut epithelial cells have been observed in rainbow trout (Oncorhyncus mykiss) (15), carp (Cyprinus carpio) (16), sea bass (Dicentrarchus labrax) (17), and several other teleost species. Although some plankton-eating species such as the sea horse may have lost gut lymphoid populations during their evolution (18), these cells, named IELs, are probably present in the gut epithelium of most bony fish species. IELs prepared from the gut of sea bass expressed TCRbeta transcripts (19), and >90% of leukocytes isolated from the carp intestine were Ig-negative lymphoid cells (16). These observations suggest that fish IELs contain mainly T cells. More recently, the rearing of germfree zebra fish revealed an evolutionarily conserved gut innate response to selected bacteria but did not target the IEL population (20). In fact, the immune status and functional capacities of fish IELs are still largely unknown.

Assuming that fish IELs consist mainly of T lymphocytes, the rainbow trout was a relevant model for further study of their diversity and responsiveness because a CDR3 length spectratyping methodology (21) has recently been developed in this species (22). This approach showed that the TCRbeta repertoire was diverse and polyclonal in spleen and pronephros of "naive" individuals, i.e., in fish that had not been subjected to clinical disease or intentional immunization. Public and private T cell responses elicited by a rhabdovirus infection or by genetic immunization were also identified through a systematic survey of VbetaJbeta spectratypes. Profiles corresponding to Vbeta4+ TCR were especially altered after viral infection, and a strong Vbeta4Jbeta1 response was systematically observed and could be considered a public response (22, 23). As six new Vbeta families were identified they were integrated into the spectratyping strategy, leading to a rather extensive description of the TCRbeta repertoire (24). In addition, several rainbow trout T cell markers have been previously published as CD8 (25) or were present in expressed sequence tag databases and could be used to further characterize the IEL phenotype.

In this work we show that IELs from the rainbow trout gut epithelium contain a highly diverse population of {alpha}beta T cells. In this respect, rainbow trout IELs were drastically different from those of their mammalian counterparts. No distinctive homing to the gut could be demonstrated for trout IELs. After infection with the viral hemorrhagic septicemia virus (VHSV), we identified modifications of the T cell repertoire of IELs similar to the modifications that we previously described for the spleen. Thus, {alpha}beta IELs shared many characteristics with those of other peripheral {alpha}beta T cell populations of rainbow trout.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Fish, virus, and infections

Rainbow trout were raised in the experimental fish facilities of the Institut National de la Recherche Agromique (Jouy-en-Josas, France). They were considered as naive when they had never shown clinical signs of disease and had not been intentionally immunized. The trout were fed normally with pellets classically used in salmonid farms and facilities. Fish were killed by overexposure to 2-phenoxyethanol diluted 1/1000. Relevant organs (thymus, pronephros, spleen, and gut) were removed aseptically for leukocyte preparations. For trafficking experiments, 4-year-old (500 g) rainbow trout from the heterozygous "clone" EQ18 (24) were used. For experimental infection, a first group of 10 fish (2 years old; 150 g) was first i.m. injected with 1–2 x 106 PFU of VHSV 25–111, an attenuated variant of strain 07-71, and was subjected to second immunization with the same virus (8–9 x 106 PFU/fish) 28 days later. A control group (mock infected) was kept in the same water. On day 49 postinfection the fish were sacrificed; RNA and cDNA were prepared for spleen and intestinal IELs, and then TCRbeta repertoires were analyzed using the immunoscope methodology. Another group of fish of the same origin was kept in the same conditions and studied in parallel as negative control for the VHSV infection.

Histology and electron microscopy

Gut samples were fixed in Bouin’s fluid and embedded in paraffin. Serial sections (5 µm) were stained with either H&E or Cleveland-Wolf trichrome. For electron microscopy, cells were fixed for 1 h in 1.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (pH 7.4), then treated with 1% osmium tetroxide, and embedded in Epon after dehydration. Thin sections stained with uranyl acetate and lead citrate were observed under an EMC12 Philips transmission electron microscope at 80 kV.

Isolation and purification of gut IELs and other leukocytes

The whole intestine was flushed with RPMI 1640 medium to remove fecal content, and adherent fat tissues were peeled off. Both small and large intestines were opened longitudinally and transferred into a 30-ml tube containing 20 ml of cold RPMI 1640. After 30 min at 0°C, the tube was shaken gently for 2 min. The shaking was repeated six to eight times to harvest all accessible cells. Collected fractions were pooled and filtered twice through nylon filters (140 µm and then 70 µm) to remove mucus-producing cells. Cells were finally recovered by centrifugation (200 x g for 20 min). For an additional step of purification, cells could be layered upon a discontinuous (67–40%) Percoll gradient and centrifuged (600 x g, 20 min) at 4°C. IELs were collected from above the 40% Percoll layer. Cells were then layered upon a 6% BSA cushion and centrifuged at 100 x g for 5 min, leading to an IEL pellet. Leukocytes from pronephros, spleen, and thymus were extracted by pressing tissues through a stainless steel grid in cold serum-free RPMI 1640 medium. Leukocytes were further isolated by centrifugation through a Ficoll gradient (lymphocyte separation medium, density 1.077; Eurobio), and used immediately for RNA preparation. The viability of cells harvested using this mechanical extraction was usually ~95% (90–97%), allowing flow cytometry analysis and RNA preparation.

Flow cytometry

Flow cytometry analyses were conducted using a standard fluorescence-activated cell analyzer (FACSCalibur). Leukocytes were incubated (30 min at 4°C) with the mouse anti-trout IgM mAb 1.14. FITC-labeled anti-mouse IgG Abs (Biosys) were used as a secondary reagent for detection. Data were analyzed with CellQuest software.

Semiquantitative PCR assay for T and B cell-specific markers

Total RNA from IEL, spleen, thymus, and muscle was reverse transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen Life Technologies). All primers for specific amplification of relevant markers are indicated in Table I. Primers were as described in Ref.25 (CD8) or were designed from the sequences of CD28 (GenBank accession number AY789435), CD4 (GenBank accession number AY973028), CD3{epsilon} (GenBank accession number CX253830), TCR{zeta} (GenBank accession number CA344798), and TCR{gamma} (GenBank accession number BX079291). The expression of Ig transcripts was studied using a primer specific for several well-expressed VH segments (for example VH1.1) in combination with a primer specific for Cµ1 sequence (CH1), leading to amplification of both Igµ and Ig{delta} H chains. Primers VB1 and CB2 were used for the amplification of TCRbeta in Fig. 2B. The amount of cDNA was normalized on the basis of beta-actin expression. PCR conditions were 94°C for 3 min and then 94°C for 45 s, 57°C for 30 s, and 72°C for 1 min for 28–35 cycles, and the amplification was stopped in the exponential phase of amplification.


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  		Table I. Primers for semiquantitative RT-PCR and immunoscope analysis

 

Figure 2
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  		FIGURE 2. Phenotypic characterization of rainbow trout intestinal IELs. A, IgM expression at the surface of intestinal IEL prepared from rainbow trout intestines. Cells were stained with the anti-trout IgM mAb 1.14 and analyzed by flow cytometry. Spleen leukocytes (S) were used as a positive control for IgM expression. B, Semiquantitative RT-PCR assay for the expression of CD8, CD4, CD28, CD4REL, CD3{epsilon}, TCR{zeta}, TCR{gamma}, TCRbeta (Vbeta1Cbeta), VH1.1-CH1, and VH6-CH1 Ig transcripts in rainbow trout IEL, thymus (T), spleen (S), and muscle (M). The amount of cDNA template was normalized on the basis of beta-actin expression, and a negative control (C) was systematically performed.

 
CDR3 length spectratyping and statistical analysis

The immunoscope analysis methodology developed for mouse or human (21) was adapted for rainbow trout, using primers specific for trout Vbeta (VB1 to VB10), Jbeta (JB1 to JB8), and Cbeta (CB1 and CB2) sequences. Immunoscope analysis was performed essentially as described in Ref.22 . Briefly, PCR was performed on the relevant cDNA using Vbeta- and Cbeta-specific primers, which amplify sequences with a given Vbeta, but with different CDR3 and Jbetas. In a second step, VbetaCbeta PCR products were subjected to runoff reactions with fluorescent Cbeta- or Jbeta-specific primers. Runoff products were loaded onto a polyacrylamide sequencing gel and size separated using an ABI 373 automated sequencer (Applied Biosystems). CDR3 length distributions were analyzed using the Immunoscope software. We used the ISEApeaks software package (2000–2002; Institut Pasteur, Paris, France) (26, 27) to extract and analyze spectratype data for relevant VbetaJbeta combinations. Perturbations between spectratypes were calculated as the average of subtraction result of the relative areas for corresponding peaks. VbetaJbeta perturbations range from 0 for identical profiles to 100 for completely different profiles. Repertoire perturbations were computed for each Vbeta as the average of perturbations for all corresponding VbetaJbeta combinations and compared between different organs or immune contexts using the nonparametrical two-tailed Mann-Whitney U test. Statistical significance was defined as p < 0.05 (*; Table III) or p < 0.01 (**; Table III). To further assess similarity between samples, hierarchical clustering with Euclidean distance was used. The complete linkage method was chosen to find similar clusters. Samples were encoded by the values of perturbations for all VbetaJbeta combinations (i.e., 32-tuples for VbetaJbeta combinations with Vbeta1–4 and Jbeta1–8). Complete clustering was performed with R software, version 1.01 (http://www.R-project.org).


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  		Table III. Results of a two-tailed Mann-Whitney test between two series of means of perturbations for each Vbeta familya

 
TCRbeta junctions

Relevant VbetaJbeta junctions were PCR from cDNA using the corresponding VB and JB primers. PCR products were cloned into the TOPO-TA cloning system (Invitrogen Life Technologies), and colonies selected at random were grown overnight in Luria-Bertani broth with ampicillin. Plasmids were purified using NucleoSpin kit (Macherey-Nagel) and sequenced.

Short-term homing

To study rainbow trout intestinal IEL, thymocyte, spleen, and pronephros leukocyte trafficking, a fluorescent assay was performed. Leukocytes (1 x 107/ml) were prepared in PBS and stained with 1 mM CFSE for 30 min at 8°C under soft agitation. Cells were washed twice with cold PBS and resuspended in PBS before injection. CFSE-stained IELs, thymocytes, splenocytes and pronephros leukocytes (1 x 107 cells) were injected i.v. or i.p. into different naive recipient rainbow trout. All of the recipient fish were sacrificed 24 h after the transfer, and cells from all lymphoid organs were isolated and analyzed by FACS.

Intestinal microflora

DNA was isolated from fecal samples collected from the gut of sacrificed fish using bead beating with glass beads and then the QIAamp DNA stool mini kit (Qiagen). Specific primers S-Eub-0339-a-A-20 and S-Univ-1385-b-A-18 (see Table I) were used to amplify bacterial 16 S rDNA (28). PCR products were cloned into the TOPO-TA cloning system (Invitrogen Life Technologies), and plasmids from colonies chosen at random were sequenced. From each individual, 16S sequences were classified into sets with <2% divergence. Each set was represented by a single representative clone corresponding to an operational taxonomic unit (OTU). These OTUs were used as reference sequences to define phylogenetic groups according to the Ribosomal Database Project II web site (http://www.cme.msu.edu/RDP/).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Rainbow trout IEL have phenotypic characteristics and markers of T cells

Histological section of rainbow trout gut showed that small round cells similar to mammal IELs were present in the lamina epithelialis interspersed with gut epithelial cells of the mucosa (Fig. 1A). Rainbow trout gut was subjected to repeated mechanical treatments and washing to extract these cells from the mucosa as in protocols classically used for the preparation of mouse IELs. During this process, most of the lamina epithelialis was taken out, except for the inner parts of intestinal villosities. A careful examination of histological sections after the extraction process clearly showed that the lamina propria remained undamaged (Fig. 1B). Cells collected from rainbow trout gut by mechanical extraction were filtered to remove mucus and epithelial cells. Occasionally, cell debris was removed by an additional centrifugation through a 6% BSA cushion. Collected IELs displayed typical features of small-sized lymphocytes with a large nucleus (Fig. 1C). These IELs were homogeneous regarding their shape and internal complexity (Fig. 2A, left panel). In flow cytometry, they appeared as a small lymphocyte population, smaller than typical monocytes and with a lower complexity than granulocytes. These cells were therefore considered as rainbow trout IELs.


Figure 1
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  		FIGURE 1. IELs of rainbow trout. A, The apical surface (mucosa) of the intestine forms the lamina epithelialis, a continuous layer of enterocytes and mucous cells overlaying a loose connective tissue, the lamina propria. Free IELs (arrows) are located between the enterocytes. Sections were stained by H&E. Bar = 30 µm. B, Histological aspect of the intestinal mucosa after IEL mechanical extraction. The major part of the lamina epithelialis has been detached from the subjacent lamina propria, which remains undamaged. Cleveland-Wolf stain was used on the paraffin section. Bar represents 200 µm. C, Electron micrograph of IELs collected by mechanical treatment. Bar represents 15 µm. Abbreviations used are: LE, lamina epithelialis; LP, lamina propria; LUM: lumen; SC: striatum compactum; M, muscularis.

 
To determine whether IELs prepared from the rainbow trout gut expressed surface Ig, they were stained using the anti-trout IgM mAb 1.14, and the proportion of IgM+ cells was measured by flow cytometry (Fig. 2A). Less than 2% of IELs were stained for IgM in contrast to the spleen, where 20–50% of leukocytes were positive. In keeping with this finding, the expression of VHDHJHCµ Ig transcripts, normally restricted to the B cell lineage, was indeed observed in the spleen but neither in thymus nor in IEL, confirming the result of anti-IgM mAb staining. PCR amplification with primers specific for different VH segments (VH1.1 and VH6) gave the same results (Fig. 2B). Confronted by the lack of reagents suitable for the identification of T cells by flow cytometry, we further studied the expression of selected T cell markers by semiquantitative RT-PCR. Amplifications using primers specific for the different TCR Vbeta segments in combination with a Cbeta-specific primer showed that TCRbeta transcripts were highly expressed in the IEL, as in the thymus or in the spleen (Fig. 2B). To confirm the presence of T cells within the IEL compartment, rainbow trout transcripts highly similar to several mammalian T cell-specific genes were also selected and their expression studied. Specific primers were designed from rainbow trout CD8 (25), CD28, and CD4 sequences or from expressed sequence tags similar to those of mammalian TCR{gamma}, TCR{zeta}, and CD3{epsilon}. When subjected to BLASTp analysis, the open reading framesf encoded by these rainbow trout sequences showed high similarity to their mammalian counterparts, indicating that they most probably constituted their true orthologs (Table II). No amplification was observed in the muscle, which was consistent with the assumed T cell-specific expression of these transcripts. They were all detected from the thymus and the spleen, which was consistent with a T cell expression pattern. All of these transcripts were also expressed in IELs, further suggesting that they probably correspond to either {alpha}beta or {gamma}{delta} T lymphocytes. These observations strongly suggested that rainbow trout IELs, like mouse or human intestinal IELs, contained a significant proportion of {alpha}beta T cells.


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  		Table II. BLASTp analysis of open reading frames encoded by rainbow trout sequences

 
Rainbow trout IELs display a highly diverse TCR repertoire

A typical feature of the human or mouse {alpha}beta IEL repertoire from the gut mucosa is a strong oligoclonality. The origin of this restricted diversity remains poorly understood but is most probably related to selective pressures, because the neonatal repertoire is polyclonal. To perform a comprehensive study of the TCRbeta repertoire of rainbow trout IELs, we used a CDR3 spectratyping method. VbetaDbetaJbetaCbeta sequences were amplified from IEL cDNA using Vbeta family-specific primers (VB1 to VB10) in combination with a Cbeta-specific primer (CB2) as previously described (22, 24). Each VbetaCbeta product was subjected to runoff using an internal Cbeta-specific primer (CB1) or Jbeta-specific primers (JB1 to JB8) for a better description of TCRbeta diversity. Typical bell-shaped profiles were systematically obtained for all VbetaJbeta combinations from naive adult fish, as shown in Fig. 3. Profiles consisted of 5–8 peaks separated by intervals of three nucleotides, corresponding to the sizes of in-frame transcripts. These results indicated that the repertoire of TCRbeta transcripts from rainbow trout IELs is highly diverse and polyclonal, like that observed for T cells from spleen or pronephros. The two sets of Vbeta6Jbeta profiles correspond to long and short forms of Vbeta6DbetaJbetaCbeta transcripts, due to an additional splicing in the Vbeta6 domain (24). Similar CDR3 length profiles were observed from IELs with spliced and unspliced transcripts for a given Vbeta6Jbeta combination, as seen previously for spleen T cells (24 ).


Figure 3
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  		FIGURE 3. Intestinal IEL TCRbeta repertoire of naive rainbow trout. cDNA from IELs were amplified using VB1-CB2 to VB10-CB2 primer combinations. Runoff products were performed using fluorescent JB or CB1 primers, separated on an automated sequencer, and analyzed with the Immunoscope software to obtain CDR3 length profiles for all VbetaJbeta or VbetaCbeta combinations. Each profile displays a bell-shaped aspect, indicating a typical diverse and polyclonal repertoire.

 
Comparing spleen and IEL TCRbeta repertoires in different individuals, we could not identify any significant difference in the average TCRbeta CDR3 length, the number of peaks, or the profile shapes for a given VbetaJbeta combination. Interestingly, the specific shape of some VbetaJbeta profiles, a "signature" slightly and reproducibly different from a Gaussian profile, was observed in both spleen and gut populations. Four Vbeta families, which are the most expressed and best studied (24), were selected for in-depth investigations. A statistical analysis was performed for an objective peak-to-peak comparison of the TCRbeta repertoire between IELs and splenocytes. Each peak area was extracted from all spectratypes with the ISEApeaks software, leading to a new data set from which statistical computations could be performed between relevant groups. We compared the perturbations (i.e., the divergence) of TCRbeta spectratypes between IEL and spleen T cells. No significant difference could be detected for any Vbeta family (Table III, group 1). These results therefore indicated that intestinal IEL contained a population of {alpha}beta T lymphocytes bearing a highly diverse available T cell repertoire in the naive rainbow trout, in contrast to what is known for different species of mammals.

Rainbow trout IELs do not traffic back to the intestine

A typical characteristic of human and mouse IELs is their homing capacity to the gut epithelium. To test the trafficking properties of rainbow trout IELs, these cells were isolated from the gut mucosa, thymus, spleen, and pronephros and stained with CFSE, and 107 cells were transferred by i.v. injection into a host with the same genetic background. For these experiments, heterozygous clones of rainbow trout were used to avoid possible artifacts due to tissue incompatibility. The homogeneity of cloned fish was checked by the analysis of six microsatellite markers in ten individuals and by MHC sequencing from three animals. Twenty-four hours after transfer, leukocytes were isolated from gut mucosa, thymus, spleen, and pronephros, and the presence of CFSE-labeled cells was assessed by flow cytometry. Three transfer experiments led to the same pattern, and typical results are shown in Table IV. Although stained lymphocytes from donor spleen and pronephros were retrieved from recipient spleen and pronephros, no fluorescent cells at all could be retrieved from the IELs of recipient trout, whatever the origin of the injected cells. The same pattern was also observed three days after injection (data not shown). To check that this distribution was not due to the delivery route of CFSE-stained cells, we also injected CFSE-stained leukocytes into the peritoneum. As for the i.v. delivery, no fluorescent cell could be detected in the IEL population of recipient fish after transfer of pronephros leukocytes, splenocytes, or IELs (data not shown). Thus, we could not observe significant difference between IEL and other peripheral leukocyte populations regarding a specific trafficking to the gut mucosa, in contrast with what was observed in mammals.


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  		Table IV. Detection of CFSE-labeled leukocytes by FACS analysisa

 
Rainbow trout IELs are responsive to a systemic viral infection

To determine whether {alpha}beta IELs can contribute to specific T cell responses against pathogens, we monitored modifications of the IEL TCRbeta repertoire after infection with VHSV a fish rhabdovirus. For IELs, as well as for spleen leukocytes, biased VbetaJbeta profiles were identified after VHSV infection, whereas all spectratypes remained bell shaped and unchanged in the control group. This observation strongly suggested that the trout gut epithelium contained responsive {alpha}beta T cells like those in spleen or pronephros. A systematic comparison of spectratypes was performed between IELs and splenocytes for each VbetaJbeta combination and identified several situations. For some combinations a bias was detected only in the spleen, as for Vbeta2Jbeta1, or only in IELs, as for Vbeta4Jbeta3 (Fig. 4). Similar modified profiles with the same amplified peak(s) were also observed for other combinations such as Vbeta4Jbeta6. Lastly, TCRbeta CDR3 length profile remained unchanged and bell shaped for many combinations like Vbeta1Jbeta7, which did not participate in the T cell response against VHSV. Profiles clearly modified for IELs after virus infection included Vbeta1Jbeta5, Vbeta1Jbeta8, Vbeta3Jbeta7, Vbeta4Jbeta1, Vbeta4Jbeta2, Vbeta4Jbeta3, Vbeta4Jbeta4, Vbeta4Jbeta6, Vbeta4Jbeta7, and Vbeta4Jbeta8. As we noticed previously from our study of the spleen T cell response against VHSV, most of the biased combinations corresponded to Vbeta4-bearing T cells. Biased profiles were also observed for VbetaJbeta combinations involving Vbeta5–10 (data not shown). These observations suggested that IEL contain {alpha}beta-responsive T cells.


Figure 4
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  		FIGURE 4. TCRbeta spectratypes of naive and VHSV-infected rainbow trout. Selected Vbeta-Cbeta spectratypes observed in spleen leukocytes and intestinal IELs of naive individuals (A) or individuals after VHSV infection (B) are shown. Amplification of cDNA from spleen leukocytes or intestinal IELs was performed using VB1CB2, VB2CB2, and VB4CB4 primer combinations and runoff synthesis was performed using fluorescent JB1, JB3, JB6, and JB7 primers.

 
Statistical analysis of TCRbeta repertoire perturbations induced by virus infection

To further validate the reality of VHSV-induced modifications in the TCRbeta repertoire of IELs after secondary infection, we subjected the spectratypes obtained for all Vbeta1–4/Jbeta1–8 combinations to a systematic numerical analysis (Table III). For each comparison, an average control profile was computed using the ISEApeaks software as the mean area for each VbetaJbeta combination on spectratypes from the considered noninfected group. Thus, control group for groups 1 and 3 of Table III was computed from the spleen repertoires of four naive individuals, whereas the control group for group 2 (Table III) was based on all repertoires from naive animals, four spleen repertoires, and three IEL repertoires, and the control group for group 4 was based on three IEL repertoires from naive animals. Perturbations between VHSV-infected individual and the control group were then computed for each peak of VbetaJbeta spectratype as differences to the control group. The resulting data set of perturbation values was subjected to statistical analysis. For each Vbeta segment (Vbeta1–4), naive tissues (IEL plus spleen) were compared with infected tissues (IEL plus spleen) (Table III, group 2). Comparison was also performed between infected and naive spleen cells (Table III, group 3) or between infected and naive IELs (Table III, group 4). In all comparisons between naive and infected repertoires, the Vbeta4 repertoire was significantly disturbed, confirming a major implication of Vbeta4 TCR into the IEL response against VHSV, as previously observed in the spleen (22, 23). To confirm this result, a complete hierarchical clustering was performed using Euclidean distance (Fig. 5). For each Vbeta segment, each condition was encoded as an 8-tuple corresponding to the perturbation of respective Vbeta/Jbeta1–8 combinations. Clustering algorithm was applied to the 8-tuple data set to assort samples into k groups without prior knowledge of group composition. For k = 3, groups containing either naive samples (sector 1) or infected samples (sectors 2 and 3) could be roughly reconstituted for the Vbeta4 family. For other Vbeta families, it was not possible to distinguish groups mainly containing either control or infected samples, confirming further the predominance of Vbeta4Jbeta rearrangements in the response to VHSV, as previously observed for spleen T cells.


Figure 5
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  		FIGURE 5. Statistical analysis of perturbations of spleen and IEL VbetaJbeta spectratypes. Schematic representation of sample clusters obtained with complete hierarchical clustering on each Vbeta family (1–4) with k = 3. VbetaJbeta perturbation of each sample was analyzed without prior knowledge of group composition.

 
Virus-biased junctions are similar in IELs and systemic lymphocytes

To analyze further the Vbeta4 response to the virus, we amplified and cloned two PCR products corresponding to representative junctions, Vbeta4Jbeta1 and Vbeta4Jbeta3, from IEL cDNA of infected trout. The Vbeta4Jbeta1 junction was especially relevant, because it was previously involved in the public response to the VHSV glycoprotein. Different Vbeta4Jbeta1 junctions were present more than once, suggesting that virus induced a Vbeta4Jbeta1 T cell response (Fig. 6). The 8-aa-long SSGDSYSE and TTGSSYSE junctions were present five and three times respectively among 15 8-aa-long clones. This observation was consistent with the corresponding Vbeta4Jbeta1 Immunoscope profile, where the peak corresponding to 8-aa-long CDR3 was clearly expanded. The expansion of the SSGDSYSE junction confirmed that the IEL response was indeed specific for the VHSV, because it had been previously characterized as the typical junction amplified in the public response to the virus. As expected from the profile shape, repeated Vbeta4Jbeta3 junctions with a length of 7- and 8-aa long were also identified. Taken together, these findings strongly suggested that IELs contained VHSV-responsive T cells.


Figure 6
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  		FIGURE 6. Vbeta4Jbeta1 and Vbeta4Jbeta3 junction sequences from intestinal IELs after VHSV infection. Vbeta4Jbeta1 (A) and Vbeta4Jbeta3 (B) junctions were amplified from gut IEL cDNA of infected fish and cloned, and randomly selected clones were sequenced. Amino acid sequences were sorted according to their CDR3 length.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In teleosts, cells with lymphoid morphology have been observed in the lamina epithelialis of rainbow trout and other teleosts, suggesting that fish do have IELs that could play an important role in gut immunity (15, 16, 17). IELs purified from rainbow trout gut epithelium indeed constituted a rather homogeneous population of small round cells with typical lymphocyte morphology, which was consistent with an IEL population counting primarily T cells. Indeed, Igµ/Ig{delta} transcripts were not detected from IEL, confirming that this population did not contain IgM+ B cells, as suggested by flow cytometry. Also, Ig{delta} or Ig{tau} transcripts were not significantly expressed in IELs (data not shown) (29). In contrast, trout IELs expressed mRNA coding for the homologs of T cell markers CD8, CD4, CD28, CD3{epsilon}, TCR{zeta}, TCR{gamma}, and TCRbeta, as did trout thymocytes and spleen leukocytes. These genes displayed high similarity with their respective mammalian counterparts and most likely constituted true orthologs. Finally, trout IEL expressed both {gamma} and beta TCR transcripts. Taken together, these observations suggested that rainbow trout IELs were mostly T cells as in human and the mouse.

Most surprising was the diversity of the TCRbeta repertoire of IELs from naive adult fish. We observed bell-shaped CDR3 TCRbeta spectratypes with 6–10 peaks for all VbetaJbeta combinations in IELs from either young or adult sexually mature fish. These observations indicated that the TCRbeta repertoire of naive rainbow trout IELs was highly diverse and polyclonal, as in pronephros and spleen TCRbeta repertoires. This finding was in sharp contrast with the situation described in mammals, where a common characteristic of all populations of gut intraepithelial {alpha}beta T cells was a strongly restricted TCRbeta diversity (6, 7, 8). A selective mechanism for the restriction of the TCRbeta diversity is generally accepted, because human infants and young rats have a diverse TCRbeta repertoire (9, 10). Similar observations were also reported in the chicken (11). In mammals and birds, the IEL TCRbeta repertoire thus becomes more and more restricted during the early life due to the selection and amplification of a few T cell clones. These observations suggested that the selective constraints shaping the TCRbeta repertoire of IEL could be different in teleosts and higher vertebrates. Because our VB1–10 primers were family-specific primers, a buffering effect may have concealed oligoclonal expansions restricted to one singular Vbeta segment belonging to a family with several members. However, this phenomenon could only affect a minority of Vbeta segments, and the magnitude of these perturbations could not match the strong oligoclonality reported in mammals. Indeed, even VbetaCbeta spectratypes that integrate all VbetaJbeta rearrangements with a given Vbeta displayed only one or a few peaks in the mouse (30). In addition, we could not find any significant difference between spectratypes from the anterior and posterior part of the gut in a given individual.

Although the TCRbeta repertoire of trout IELs was highly diverse in naive individuals, expanded clones were found in the gut mucosa during the response to a systemic infection. We have previously shown that VHSV infection induces significant and specific modifications of the TCRbeta repertoire, corresponding to public and private T cell responses. Vbeta4Jbeta1 rearrangements were amplified among spleen T cells in response to the viral glycoprotein and were observed in fish from different origins (22). In the present study, we showed that significant modifications of the IEL TCRbeta repertoire were also induced by a systemic viral infection. As previously observed in the spleen, the most evident perturbations involved Vbeta4 rearrangements. Sequencing of cloned VBJB PCR products corresponding to spectratypes with reduced numbers of peaks, such as Vbeta4Jbeta1 and Vbeta4Jbeta3, identified recurrent junctions corresponding to expanded clones. Interestingly, the Vbeta4Jbeta1 junction SSGDSYSE, which was the most expanded in the spleen public T cell response to the VHSV glycoprotein (23), was also amplified in IELs from infected fish. These observations strongly suggested that IELs contain virus-responsive T cell clones.

Rainbow trout intraepithelial and spleen {alpha}beta T cells could not be distinguished by either morphological or phenotypic characteristics or by their TCRbeta repertoire in healthy or virus-infected fish. Moreover, rainbow trout IEL did not seem to specifically traffic to the gut epithelium after adoptive transfer. This was clearly different from the trafficking behavior of IELs described in mammals (31, 32, 33). Although we recovered only ~1% of transferred labeled lymphocytes, we consistently observed them only from spleen and pronephros but never from the gut mucosa whatever their origin (pronephros, spleen, or gut mucosa). All of these findings suggest that teleost fish may lack a discrete {alpha}beta IEL compartment with specific properties, as reported for several species of higher vertebrates. In this perspective, fish T cells would enter the gut epithelium without specific requirements and would therefore display the general characteristics of systemic {alpha}beta T cells. We are conscious that our spectratyping methodology delivers only a global view of the TCRbeta repertoire of rainbow trout. Thus, we cannot exclude the possibility that a minor T cell subset with oligoclonal diversity may be present in the gut epithelium. However, our results clearly indicate that the main part of {alpha}beta IELs share the same phenotypic, homing, and TCRbeta repertoire properties with systemic T cells. In mouse, it has been proposed that thymus-dependent blast progenitors are stimulated in Peyer’s patches or mesenteric lymph nodes, pass to the thoracic duct, and migrate from the lymph to the blood from which they seed the lamina propria and then the epithelium of the whole intestine (34). Gut mucosal lymphocytes are able to recirculate upon stimulation with cognate Ag. Successive stimulations therefore lead to new rounds of T cell trafficking and to a strong amplification of selected clones, shaping an oligoclonal IEL repertoire. In the absence of specialized lymphoid structures associated to the gut, the progenitors of trout IELs may lack the stimulation sites that are necessary for the successive and regular reactivation/trafficking cycles induced by their cognate Ags. Alternatively, the gut antigenic environment of rainbow trout may not provide the restimulation signals required for a progressive narrowing of the repertoire. The gut microflora probably constitutes an important source for IEL stimulation. In the fish, the use of molecular inventory methods revealed that zebra fish (20) and salmonids (35, 36) harbor a specific and rather diverse gut microbiota that is comparable to what was observed in the human gut (28). To investigate the structure of the gut microbiota of the rainbow trout from our fish facilities, we sequenced libraries of bacterial 16S rRNA genes amplified from the DNA of gut samples of three individuals kept in the same aquaculture conditions (data not shown). In average, 13 OTUs were specific to one individual, and only Bacillus cereus and Clostridium gasigenes were found in common to the three individuals. With a total of 34 OTUs, these observations were consistent with observations previously reported for fish microflora, indicating that the polyclonal repertoire of {alpha}beta IEL could not be explained by the paucity or the odd structure of the gut microflora.

In conclusion, this report reveals that rainbow trout IELs contain T cells with the same phenotypic and functional characteristics as systemic T cells. Thus, fish {alpha}beta IELs may essentially correspond to a random sample from the systemic {alpha}beta T cell population. As the features of gut microbiota seems to be rather equivalent in humans and fish, the large TCRbeta diversity of trout IEL is probably due to the distinctive anatomy of GALT in fish, which would not provide the appropriate microenvironments for T cell stimulation and selection. The same line of explanation was finally accepted for the lack of efficient somatic mutation in fish and amphibian immunoglobulins when all enzymatic pathways and cellular components were available (37). Further studies will be necessary to fully elucidate the origin and functions of fish IELs, which would provide interesting clues about the evolution of mucosal immunity and could improve the efficiency of oral fish vaccines.


   Acknowledgments
 
We thank John D Hansen for trout CD4 primers and Dominique Buzzoni-Gatel for discussions about homing experiments. Simon Fillatreau, Jean Kanellopoulos, and Louis du Pasquier are also gratefully acknowledged for their comments and suggestions about the manuscript.


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


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

1 This work was supported by the Institut National de la Recherche Agronomique. Back

2 Address correspondence and reprint requests to Dr. Pierre Boudinot, Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas cedex, France. E-mail address: pierre.boudinot{at}jouy.inra.fr Back

3 Abbreviations used in this paper: IEL, intraepithelial lymphocyte; OUT, operational taxonomic unit; VHSV, viral hemorrhagic septicemia virus. Back

Received for publication September 22, 2005. Accepted for publication January 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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