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Rhabdovirus Infection Induces Public and Private T Cell Responses in Teleost Fish¹

Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Many viruses induce a strong T cell response that contributes to the elimination of infected cells presenting viral peptides by MHC molecules. The structure and expression of genes encoding molecules homologous to mammalian TCRs have been recently characterized in rainbow trout and in several teleost species, but the T cell response against pathogens has not been directly demonstrated. To study the modifications of the T cell repertoire during an acute viral infection in rainbow trout, we adapted the immunoscope methodology, which consists of spectratyping the complementarity-determining region 3 length of the TCR chain. We showed that the naive T cell repertoire is polyclonal and highly diverse in the naive rainbow trout. Using viral hemorrhagic septicemia virus (VHSV), which provokes an acute infection in rainbow trout, we identified skewed complementarity-determining region 3 size profiles for several VJ combinations, corresponding to T cell clonal expansions during primary and secondary response to VHSV. Both public and private T cell expansions were shown by immunoscope analysis of spleen cells from several infected individuals of a rainbow trout clone sharing the same genetic background. The public response to VHSV consisted of expansion of V4J1 T cell, which appeared early during the primary response and was strongly boosted during the secondary response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viral hemorrhagic septicemia virus (VHSV)³ is an important viral disease in European trout farms (1). Marked hemorrhagic lesions and high mortality rates among juveniles characterize this systemic disease. The nonsegmented negative-strand RNA genome of the VHSV is now completely resolved (2). It encodes six proteins: the nucleocapsid N (3), the polymerase-associated protein P, the matrix protein M (4), the transmembrane glycoprotein G (5), the nonstructural protein NV (6), and the RNA-dependent RNA polymerase, L.

VHSV infection results in production of neutralizing Abs, which are exclusively directed to the viral membrane G protein. Neutralizing Abs certainly constitute an important element of the protection against the virus. Passive transfer of neutralizing trout polyclonal or mouse mAbs resulted in complete protection against a lethal challenge (7). More recently, DNA immunization with the cloned G gene of VHSV or infectious hematopoietic necrosis virus, a fish rhabdovirus closely related to the VHSV, showed that expression of this protein was sufficient to afford complete protection (8, 9). On the contrary, DNA immunizations with the N, P, or M genes of infectious hemopoietic necrosis virus were totally inefficient (10). Although the humoral Ab response to VHSV has been extensively studied, the mechanism underlying the establishment of the immune response is not yet clearly understood and, especially, little is known about the T cell contribution to the response. In fact, a contribution of the T cell compartment can be anticipated from the observation that the recombinant G protein produced in vitro had a poor protective activity compared with the intact virus or the DNA vaccine (11).

The reality of a T cell response in fish is suggested by the description of TCR and MHC genes in several species. The diversity of TCR chain has been first reported in rainbow trout (Oncorhynchus mykiss) (12, 13). The TCR locus of rainbow trout has been partly characterized, with a sequence description of the genomic region containing the DJC genes (14). TCR sequences are now available for several other fish species, such as horned shark (Heterodontus francisci) (15), skate (Raja eglanteria) (16), catfish (17), and sea bass (18). In addition, the sequence and polymorphism of fish MHC strongly suggest that TCR can recognize the Ag in an MHC-restricted context (19, 20, 21). In rainbow trout LMP2, LMP2/d, TAP1A, and TAP2B also have been located in the class Ia locus (20), which suggests that class I-dependent Ag processing and presentation could follow the same pathway as in mammals. In cloned goldfish, the involvement of a T cell response during viral infection was recently suggested by the observation of a specific cell-mediated lysis of virus-infected syngenic target cells (22, 23). However, the effector cells were not unequivocally identified as T cells. The importance of the T cell response during viral infections is still largely unknown, in part because of the lack of suitable T cell-specific Abs.

In this context, the monitoring of the modifications of the T cell repertoire during viral infection would be most helpful. The development of a complementarity-determining region 3 (CDR3) length spectratyping methodology (24, 25) named "immunoscope" (26) has made possible the systematic description of T cell repertoires in humans and in the mouse.

Using this methodology, which we adapted to the rainbow trout, we describe in the present report the changes of CDR3 length distributions induced by VHSV viral infection. We report altered profiles during infection with an attenuated virus, which were further biased after a subsequent challenge with a virulent strain of VHSV. As it was reported in mice (27), we observed "public" and "private" T cell responses in rainbow trout. The public response consists in TCR rearrangement reproducibly present in all individuals, whereas the private components seem to emerge stochastically and implicate different VJ combinations in different trout of the same genetic background. To our knowledge, this work provides the first direct demonstration of a T cell response against a virus in a teleost fish.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fish

Rainbow trout were raised in the fish facilities of Institut National de la Recherche Agronomique (Jouy-en-Josas, France). The so-called "INRA synthetic strain" corresponds to a rainbow trout population. It was generated from successive introductions between 1976 and 1983 of several domestic populations from the United States and France that were pooled and then maintained as a single population by random mating during four to five generations. A small number of homozygous trout were obtained by gynogenesis from the synthetic population as described by Diter et al. (28). Some of the gynogenetic animals (all females) were subjected to a treatment with methyltestosterone and developed as homozygous neomales. Rainbow trout clones were then obtained by crossing the neomales with homozygous females from a different gynogenesis experiment. Therefore, animals within each clone are heterozygous but share the same genetic background.

Immunization and virus challenge

The attenuated 25-111 variant of strain 07-71 of VHSV was used to infect fish through i.m. injection of 1–5 x 10⁵ PFU/trout. This infection usually leads to a good protection against a subsequent lethal infection. Four weeks later, fish received a second i.m. injection of variant 25-111 or were subjected to challenge with the virulent strain 07-71 (75 x 10⁶ PFU/trout).

Immunoscope

The immunoscope methodology developed for mouse or human (24, 26) was adapted for rainbow trout, using primers specific for trout V, J, and C sequences. The genomic organization of the V locus has not yet been characterized. Therefore, V family-specific primers were designed to avoid cross-hybridization among the four V families described so far. We chose the primers in the framework region (framework region-2 region for V1 and V3, framework region-1 region for V2 and V4) to amplify most of the V segments in each family. J primers were determined from the genomic sequence (14), and they were designed to be specific for each of the 10 J segments except for J4, because J2 and J4 sequences are similar. A J2–4 primer was designed in a region similar in J2 and J4 to amplify both kinds of rearrangement with the same efficiency. Two C primers were designed from the work of Partula (29) and de Guerra (30). Primers used are shown in Table I.

TCR

Immunoscope computer analysis

CDR3 length distributions were analyzed using the immunoscope software as described by Pannetier et al. (26). The Immunoscope data toolbox of ISEA peaks (A. Collette and A. Six, Institut Pasteur, Paris, France), an Excel platform (Microsoft, Redmond, VA) for GeneScan (Applied Biosystems) and immunoscope data retrieval, was used for immunoscope macro design and editing of CDR3 spectratypes.

CDR3 cloning and sequencing

To investigate the sequence composition of expanded peaks of skewed V-J profiles, we performed PCR from the relevant cDNA, using the corresponding V and J primers. PCR products were purified though Sephacryl S-400 columns (Pharmacia Biotech, Uppsala, Sweden) and cloned using the TOPO-TA cloning system (Invitrogen, San Diego, CA). Several clones were then picked at random and subjected to sequencing. Sequences were aligned using the Genetic Computer Group (Madison, WI) package, and CDR3 region was considered between residues 96 and 106 (Kabat numbering).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Primer validation for TCR immunoscope analysis in rainbow trout

The first step of validation for the "rainbow trout immunoscope" was to verify the specificity of primers. For these experiments, we used a mixture of spleen RNA from four trout of the INRA synthetic strain. This strain displays a large genetic diversity, and the results should be naturally extendable to any population of rainbow trout.

The specificity of V family-specific primers was first investigated by enzymatic digestion of V-C PCR products with restriction enzymes specific for each V family (Fig. 1A). V2C, V3C, and V4C gave restriction profiles consistent with a family-specific amplification with each V primer (Fig. 1B). The V1C product was only partially digested by EcoRI, suggesting that some V1 segments lacked the EcoRI site. This was confirmed on the basis of V1J product sequencing (see Table II).

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Specific restriction patterns of V-C PCR products. V-C amplification products obtained from spleen cDNA of naive rainbow trout were subjected to restriction analysis with enzymes giving specific patterns for each V sequence. A, Restriction map of V-C products. B, PCR products for V1-C (lines 2–5), V2-C (lines 6–9), V3-C (lines 10–13), and V4-C (lines 14–17) digested with EcoRI (3, 7, 11, and 15), KpnI (4, 8, 12, and 16), PstI (5, 9, 13, and 17), or not digested (2, 6, 10, and 14). Lines 1 and 18, Size markers (pbr322 AluI).

View this table: [in this window] [in a new window]   Table II. Specificity of V and J primers

To verify the specificity of J primers, combinations of V1 or V2 with all J primers were used to perform PCR. All combinations gave PCR products of the expected size, except those including two different J10 primers. In fact, no amplification was obtained using combinations of any V primers with the two different J10 primers, suggesting that this segment is probably not used in the fish studied. As mentioned above, the V-J PCR products were cloned and several clones were sequenced. The specificity of nine J primers was attested in that no unexpected cross-amplification was detected in the sequenced samples (see Table II). The J2–4 primer amplified J2 and J4 templates with equivalent efficiencies. Therefore, we used primers specific for J1, J2, J3, J5, J6, J7, J8, and J9 segments and primer J2–4 to amplify both J2 and J4 segments.

Immunoscope profiles in naive rainbow trout

We first performed an Immunoscope analysis of the T cell repertoire in the spleens of naive rainbow trout of the INRA synthetic strain. We used all V family-specific primers, and run-off reactions were performed with C1-fluorescent primer to assess the CDR3 length diversity and stability in different individuals. We obtained profiles composed of five to eight peaks representing pools of TCR with similar CDR3 size (Fig. 2A). The peaks were separated by 3-nt intervals, corresponding to the sizes of in-frame transcripts. Comparing different individuals, we did not detect significant variations either in the number of peaks or in the profile shape. For each V family, this experiment presents a general survey of the CDR3 length distribution. As in mammals, random nucleotide additions and deletions during the V(D)J recombination lead to a complex population of CDR3 sequences that is represented by a gaussian distribution of peaks—a spectratype. These gaussian distributions signify that the spleen T lymphocyte population is polyclonal in naive rainbow trout as in naive mice. This was already suggested by previous sequence studies on rainbow trout TCR junctions (13, 31). We performed the same analysis with trout lymphocytes obtained from pronephros and thymus. Spectratypes in the pronephros and in the thymus were similar to those obtained from the spleen (Fig. 2, B and C). These results confirmed that these organs contain large populations of T lymphocytes, corresponding to a highly diverse available T cell repertoire.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Immunoscope analysis of leukocyte populations from different lymphoid organs in naive rainbow trout reveals gaussian profiles for all V families. cDNA from the spleen (A) of two individuals, from the pronephros (B) or from the thymus (C), were amplified with V1–4 primers coupled to C2 primer. PCR products were subjected to run-off using the fluorescent C1 primer, separated on an automated sequencer, and analyzed with the Immunoscope software to obtain CDR3 length profiles. Each profile represents the CDR3 size distribution for a given V family. Fragment length is on the x-axis and fluorescence intensity is on the y-axis. V families are indicated on the top.

A group of 12 fish were injected with 5 x 10⁵ PFU of 25-111, an attenuated variant of strain 07-71 of VHSV, and were subjected to challenge with the virulent strain 07-71 on day 27. On days 1, 7, 14, 21, 27, 32, 35, and 40 postinfection, one trout was sacrificed for spleen cDNA preparation and analysis using the immunoscope methodology. A typical gaussian distribution of five to eight CDR3 length was observed on days 1, 14, 21, and 27 for all VJ combinations. By contrast, profiles were clearly altered for several VJ combinations on day 7 (V4J1, V4J3, V4J8), on day 35 (V1J5, V1J6, V1J8, V2J8, V3J6, V3J7, V4J1, V4J3, V4J5, V4J7, V4J8), and on day 40 (V1J3, V2J1, V2J7, V2J8, V3J1, V3J3, V4J1, V4J3, V4J8). These altered profiles indicated that the virus infection had induced strong modifications of the T cell repertoire. It is interesting to note that all of the bias observed early at day 7 postinfection corresponded to a V4J combination. They were also present on days 35 and 40, but not on days 14, 21, and 27. For the V4J1 combination, a bias was reproducibly and clearly observed in all infected animals: the distribution of V4J1 CDR3 size was already modified on day 7 and dramatically biased on day 40 (Fig. 3). These skewed repertoires were concomitant with the expected peaks of viral replication. In addition, the amplified peaks on days 7, 35, and 40 were all composed of CDR3 of similar length (8 aa). Taken together, these results strongly suggest that the expansion of V4J1 cells represented a specific T cell response to the virus.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Immunoscope analysis of typical V4J1 CDR3 length profiles during primary and secondary response against VHSV. Rainbow trout were infected with the 25-111 attenuated variant of the VHSV strain 07-71 and challenged on day 27 with the 07-71 strain. To obtain V4J1 CDR3 size profiles, spleen cDNA from relevant infected fish was amplified using V4 and C2 primers, and run-off was performed with fluorescent J1 primer. Run-off products on V4J1 rearrangements were separated on an automated sequencer and analyzed with the immunoscope and ISEA peaks software to obtain CDR3 length profiles for days 1, 7, 14, 27, 32, and 40. Fragment length is on the x-axis and fluorescence intensity is on the y-axis

Sequence analysis of the V4J1-biased spectratype

To verify that the altered profiles really correspond to an expansion of T cell clones, we amplified and cloned the sample corresponding to the V4J1 profile of day 40, which showed drastic expansion of a single peak. Several clones were randomly picked up and subjected to sequencing (Fig. 4). Thirteen sequences of the 25 analyzed had a CDR3 composed of 8 aa corresponding to the size of the expanded peak. Among these 13 sequences, one sequence was found six times and another three times. The other sequences were all different and were found only once in the sample. Therefore, the CDR3 distribution size had been modified by selection and expansion of two T cell clones. It is interesting to note that only one junction out of 25 corresponded to a nonproductive rearrangement. In naive rainbow trout, it was shown that approximately one-third of the TCR transcripts had out-of-frame V(D)J junctions (31).

View larger version (35K): [in this window] [in a new window]   FIGURE 4. CDR3 sequences from the V4J1 skewed profile identified in Fig. 3. Spleen cDNA from day 40 (day 13 after viral challenge) was amplified using V4J1 primers. PCR product was purified and cloned, and clones picked at random were sequenced. Nucleotide and amino acid sequences of the in-frame junctions were sorted according to their CDR3 length. Underlined nucleotides correspond to the D sequence. Boldfaced nucleotides are not germline-encoded and most probably represent N additions. Italicized nucleotides are compatible with P addition mechanism. Nucleotides that are both boldfaced and italicized can be either P or N additions. An asterisk indicates the only out-of-frame rearrangements found.

Modifications of TCR CDR3 length profiles during the VHSV infection in clones of rainbow trout

To follow up the T cell response against the virus in different individuals without the influence of fish-to-fish genetic diversity, we used cloned rainbow trout (clone EQ2). We first assessed the diversity of the TCR repertoire of four naive fish from clone EQ2. A typical immunoscope picture is shown in Fig. 5. As observed in the previous experiment, all VJ combinations amplified a highly diverse population of TCR transcripts, resulting in typical gaussian profiles. This observation confirmed that fish from clone EQ2 were appropriate for analysis of T cell repertoire modification. A group of 10 fish from clone EQ2 was infected with the 25-111 variant of VHSV and then received a second injection of the same virus on day 27. Spleen T cell repertoire was analyzed in four infected fish, two from day 41 (day 14 after challenge) and two from day 47 (day 20 after challenge). The corresponding Immunoscope profiles obtained with all V-J combinations are shown in Fig. 6. Several V-J combinations showed modified CDR3-, compared with a naive repertoire represented in Fig. 5. Some biases seemed to be recurrent: V4-J1, V4-J7, V2-J2, V2-J2, V3-J2, and V3-J3 (Table III). Moreover, the V4-J1 response was observed in all infected animals and could be considered as a true public response. The biases in V2-J2 and V4-J7, which were observed in three individuals, may also correspond to a public response. However, most of the modifications were found once or twice and may correspond to private responses appearing at random in individual fish. For each V-J combination giving a redundant skewed profile, the expanded peak is always composed of runoff products of the same size, which probably means expansion of T cells sharing the same CDR3.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Typical immunoscope analysis of V(D)J rearrangements in spleen transcripts of naive rainbow trout (clone EQ2). cDNA from spleen cells of naive fish were amplified using V1–4, C2 primer combinations. Run-off products were performed using fluorescent J or C1 primers, separated on an automated sequencer, and analyzed with the immunoscope and ISEApeaks software to obtain CDR3 length profiles for all VJ or VC combinations. The profiles are representative of four independent experiments on fish with the same genetic background.

View larger version (80K): [in this window] [in a new window]   FIGURE 6. Immunoscope profiles of infected rainbow trout (clone EQ2). Four individuals from the same clone were infected with the 25-111 attenuated variant of VHSV on days 1 and 27. Spleen leukocytes were analyzed 14 days (A and B) or 20 days (C and D) after the second infection. cDNA from spleen cells were amplified using V1–4, C2 primer combinations. Run-off products were performed using fluorescent J or C1 primers, separated on an automated sequencer, and analyzed with the Immunoscope and ISEA peaks software to obtain CDR3 length profiles for all VJ or VC combinations.

View this table: [in this window] [in a new window]   Table III. V-J skewed combinations during response to VHSV

TCR CDR3 sequence of T cells amplified during infection in different individuals of the same clone

We further analyzed V4-J1 profiles corresponding to the public response identified in cloned trout (Fig. 7). We amplified and cloned these V4-J1 rearrangements from spleen cDNA of two infected trout sacrificed on day 41 (trout 1) and on day 47 (trout 3). Twelve clones of 21 in trout 1 and 13 of 20 in trout 3 had a CDR3 sequence of 8 aa, corresponding to the size of the expanded peak. In both fish, the same junction was highly represented: 10 of 12 clones in trout 1 and 13 of 13 in trout 3. Thus, the V4-J1 public response conserved not only the usage of the V4-J1 combination and the CDR3 length, but also the CDR3 sequence. All other junctions were obtained only once. In a control experiment, V4-J1 PCR products from a nonimmunized fish were similarly amplified, cloned, and subjected to sequencing. All 56 clones showed different CDR3- sequences (data not shown). Therefore, we assumed that the redundant CDR3 sequences correspond to a virus-specific clonal expansion of T cells. To further analyze a private response, V3-J7 junctions from trout 3 were similarly amplified and cloned. Sixteen clones were sequenced, of which eight had CDR3 sequences of 8 aa, corresponding to the expanded peak. Of these eight clones, five showed the same CDR3 sequence. Thus, only one V3-J7 sequence was amplified, as was the case for the V4-J1 combination analyzed in the same context. All other sequences were found only once, as previously noted for V4-J1. These results definitely establish that the V3-J7 skewed profile reflects a clonal T cell expansion and strongly suggest that all other biased profiles should be considered in the same way.

View larger version (59K): [in this window] [in a new window]   FIGURE 7. CDR3 sequences of the V4J1 (A) and V3J7 (B) rearrangements after second infection (public and private responses, respectively). Spleen cDNA from infected cloned fish was amplified using V4-J1 primers (fish 1 and 3) or V3-J7 primers (fish 3). PCR product was purified and cloned, and clones picked at random were sequenced. Nucleotide and amino acid sequences of the in-frame junctions were listed by their CDR3 length. Underlined nucleotides correspond to the D sequence. Boldfaced nucleotides are not germline-encoded nucleotides and most probably represent N additions. Italicized nucleotides are compatible with P addition mechanism. Nucleotides that are both boldfaced and italicized can be either P or N additions. An asterisk indicates the only out-of-frame rearrangements found.

TCR

VDJ junction analysis at the nucleotidic level

Thirty-six different V4-J1 and 12 different V3-J7 CDR3 sequences were identified in this work (Figs. 4 and 7). Most of these junctions had a recognizable D segment (31 of 36 V4-J1 and 10 of 12 V3-J7, respectively). Seven junctions showed no D segment. One junction from trout 3 (Fig. 7) could encompass two D segments, which would mean the existence of at least two D genes. Because the genomic sequences of V segments are not known, it was not always possible to decide unequivocally what nucleotides came from V segments and which represented N additions at the VJ junction. On the contrary, the DJ junction could be analyzed unambiguously from comparison with D and J genomic sequences. All V(D)J junctions but three had N additions. Ten DJ junctions showed no N additions (20%). This value is close to that observed in the adult mouse, whereas 40% of adult rainbow trout DJ junctions previously published were without N additions (31). Contrary to mammals, there is no preferential addition of GC over AT, as previously observed in rainbow trout (31). Potential P nucleotides could be identified in five junctions. The size of the CDR3 loop varies from 6–13 residues (mean, 8.3), which corresponds to a longer size than the mean previously published for rainbow trout (7.2 compared with 9.9 in the adult mouse). This result could correspond to special constraints of the two analyzed rainbow trout V-J combinations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this work, we directly addressed the functionality of a T cell response in a teleost fish during viral infection by monitoring the changes imposed on a polyclonal and diverse T cell repertoire. Indeed, the Immunoscope methodology allowed us to study systematically the diversity of the T cell repertoire and to identify several public and private components of the T cell response against an acute viral infection.

Graft rejection has provided the first experimental indications suggesting that teleosts should possess a functional T cell-mediated immunity (32, 33). In catfish, a mixed lymphocyte reaction was described with Ig^- lymphocytes (34). More recently, the development of catfish clonal long-term lines of B cells, T cells, and macrophages allowed the setup of in vitro assays for allospecific cytotoxicity. Several leukocyte cell lines were identified as ⁺ allospecific effectors, providing convincing evidence for the existence of cytotoxic T cells in teleost (35). Autologous specific cell-mediated cytotoxicity has been described in catfish and in goldfish. In catfish, the in vitro anti-hapten plaque-forming cell response required Ig⁺ and Ig^- lymphocytes, as well as presenting cells (36), suggesting a T and B cell cooperation for Ab production. More recently, an autologous cell-mediated response against a syngenic cell line infected with infectious pancreatic necrosis virus was described in the goldfish (Carassius auratus) (23). However, in the above studies, the cells responsible for the autologous CTL-like activity were not unambiguously characterized as ⁺ T lymphocytes.

This PCR-based strategy of CDR3 length spectratyping is more flexible and systematic than classical repertoire analysis based on random sequencing of cloned CDR3 regions. Using a set of primers for known TCR segments, we have detected clear clonal expansions for several VJ combinations. However, this may be a partial description of the T cell response. Indeed, we cannot rule out the possibility that weak expansions have been missed, because the profile of a CDR3 size distribution corresponds to the superposition of the profiles for different V members of the same family. The initial VC amplification is a competitive amplification of sequences that are identical or quasi-identical, except in their CDR3 region. Because many lymphocytes in the complex mixture of cells in the initial spleen sample share the same V, J, and CDR3 size (but have different CDR3 sequences), the patterns obtained are statistically "buffered." Consequently, a slight clonal proliferation could be more difficult to detect using V family-specific primers than using V segment-specific primers. Additionally, this description will be further enriched as new V families (or new J segments) are identified.

Immunoscope analysis of the TCR diversity in spleen, pronephros, and thymus of naive rainbow trout showed gaussian distributions of CDR3 size for all VC and VJ combinations studied. It means that these repertoires are polyclonal and not significantly skewed by recombination constraints or selective pressures. Provided that pairing follows similar combinatory rules, the diversity of the ⁺ T cell repertoire of rainbow trout therefore is basically comparable to that of mouse ⁺ T cells. Sequencing studies have already shown a large diversity of rainbow trout TCR chain (37). Thus, T cells have probably conserved their characteristics of diversity and population dynamics through the gnathostome evolution. After viral infection, we observed several VJ profiles showing a clear, unique expanded peak at a given CDR3 size. Skewed profiles were observed in different V families, despite the "buffering effect" reinforced by the usage of V family-specific primers. Thus, clonal amplifications are strong enough to be clearly detected using V family-specific primers, even for families that comprise several members. In mammals, lymphocytic choriomeningitis virus and SIV were also shown to induce strong T cell clonal expansions (38, 39, 40). The magnitude of rainbow trout T cell expansions is also attested by the low frequency of out-of-frame junctions recorded in infected compared with naive rainbow trout. In naive rainbow trout with the same genetic background, we found 11 out-of-frame junctions out of 56 V4-J1 CDR3 sequences analyzed (data not shown). This was consistent with previous studies in which nonproductive junctions represented 20–30% of transcript CDR3 sequences in naive fish (31). Most likely, the low frequency of out-of-frame junctions we observed in infected fish was caused by clonal expansion of cells expressing in-frame TCR transcripts only.

Skewed profiles were already observed on day 7 postinfection for V4J1, V4J3, and V4J8 combinations. Rainbow trout T cell response therefore appears relatively early and seems to be concomitant with the peak of VHSV replication. Furthermore, these VJ biases were no longer detected at days 14, 21, and 32, which could be interpreted as a decline in number of reactive T cell clones. Most interestingly, CDR3 of the same size were strongly expanded after the second infection, which is reminiscent of the kinetics observed in mammals for different viral infections (38, 40, 41). Actually, this is a good indication of the existence of a memory, which was never directly and clearly demonstrated in teleosts.

In cloned fish, the V1 family showed no evident clonal expansion, even though our analysis putatively encompassed both CD8⁺ and CD4⁺ T cells. Neither public nor private expansion could be observed for V1-expressing T cells, whereas several V1 segments were amplified with the V1 family-specific primer. Considering the number of potential epitopes in the virus, it is expected that T cell-expressing V gene segments from all four known families would be positively selected. However, in this experiment we investigated a secondary response against the virus, which could explain the lack of some reactive cells. Indeed, studies in the mouse have shown that the secondary effector T cell pool was much less diverse than the primary one, due to Ag-driven selective expansions of specific subpopulations (42, 43). Another explanation for this lack of V1 reactivity may be that TCR specificity is partially determined by CDR1 and CDR2 residues.

The fact that all combinations skewed during the primary response were composed of segments from the V4 family (including the V4J1 public response) probably corresponds to a higher affinity of clones using the V4 rearrangements for at least one viral dominant epitope. However, it could also result from a differential buffer effect within V families. The V4 family contains one or only a few segments, and the "buffer effect" is lower than for the other families, allowing for detection of smaller expansions in this family.

Comparing the repertoire of different infected animals, we can consider that the V4J1 expansion corresponds to a public response against one viral epitope, whereas the other expanded combinations are not observed in all fish and can be considered as private responses. The public arm of the response corresponded to the expansion of the same V4-J1 CDR3 sequence in two different individuals. This situation is in contrast with some mouse T cell responses in which different CDR3 sequences were expanded in genetically identical individuals (41). It suggests that the diversity of T cells against a given epitope may be restricted in rainbow trout, or that the selective process during the secondary response is especially powerful. Private responses are not present in all individuals either, because the available repertoire lacked the precursors or because the corresponding TCR had a low affinity for the epitope and had not been strongly selected during the secondary response. In any case, these results show that the trout T cell repertoire of anti-VHSV TCRs is large enough to allow the selection of diverse private responses using different VJ combinations in different individuals.

In conclusion, this report provides the first direct and systematic analysis of the T cell response diversity in a teleost fish. This study also shows that diversity and dynamics of immune repertoires in lower vertebrates are basically comparable to those of the mouse, justifying the usage of the Immunoscope strategy to further characterize immune systems of these species.

	Acknowledgments

 
We thank A. Collette, A. Six, and P.-A. Cazenave for their helpful discussions and expert advice and for providing the ISEA peaks software. B. Loriot and the staff of the experimental fish facilities are also acknowledged for their excellent technical assistance. Clonal fish were provided by M. Dorson and E. Quillet. We thank L. du Pasquier and J. Kanellopoulos for their comments and suggestions on the manuscript.

	Footnotes

 
¹ This work was supported by the Institut National de la Recherche Agronomique and European Community Project FAIR 98-4026.

² Address correspondence and reprint requests to Dr. Abdenour Benmansour, Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas Cedex, France. E-mail address: abdenour{at}jouy.inra.fr

³ Abbreviations used in this paper: VHSV, viral hemorrhagic septicemia virus; CDR3, complementarity-determining region 3.

Received for publication July 25, 2001. Accepted for publication September 26, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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