Murine
-herpesvirus infection causes Vß4-specific CDR3-restricted clonal expansions within CD8+ peripheral blood T lymphocytes
Charles L. Hardy1,4,
Sharon L. Silins3,
David. L. Woodland1,2,5 and
Marcia A. Blackman1,2,5
1 Department of Immunology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
2 Department of Pathology, University of Tennessee, Memphis, TN 38163, USA
3 EpsteinBarr Virus Unit, Queensland Institute of Medical Research and University of Queensland Joint Oncology Program, Brisbane, Queensland 4029, Australia
Correspondence to:
M. A. Blackman, Trudeau Institute, 100 Algonquin Avenue, Saranac Lake, NY 12983, USA
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Abstract
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Infection of mice with the
-herpesvirus MHV-68 results in lytic infection in the lung cleared by CD8+ cells and establishment of lifelong latency. An EpsteinBarr virus (EBV)-like infectious mononucleosis (IM) syndrome emerges ~3 weeks after infection. In human IM, the majority of T cells in the peripheral blood are monoclonal or oligoclonal and are frequently specific for lytic or latent viral epitopes. However, a unique feature of MHV-68-induced IM is a prominent MHC haplotype-independent expansion of CD8+ T cells, the majority of which utilize Vß4 chains in their
ßTCR. The ligand driving the Vß4 expansion is unknown, but the Vß bias and MHC haplotype independence raised the possibility that these cells were responding to a virally encoded or a virally induced endogenous superantigen (sAg). The aim of this study was to determine whether this rapidly proliferating subset is composed of polyclonally or clonally expanded T cells. Complementarity-determining region (CDR)-3 size analysis of Vß4+CD8+ cells in infected mice demonstrated CDR3-restricted expansions in the Vß4 family as a whole. More refined analysis demonstrated major distortions in every Jß subfamily. VDJ junctional region sequencing indicated that these CDR3 size-restricted expansions were composed of clonal or oligoclonal populations. The sequences were largely unique in individual mice, although evidence for `public' or highly conserved T cell expansions was also seen between different mice. Taken together with previous studies showing an apparent MHC independence, the data suggest that a novel ligand, distinct from conventional sAg and peptideMHC, drives proliferation of Vß4+CD8+ T cells.
Keywords: MHV-68, superantigens, TCR repertoire
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Introduction
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Murine
-herpesvirus-68 (MHV-68), a
2-herpesvirus, shares biological features and sequence homology with human herpesvirus 8 and EpsteinBarr virus (EBV) (1,2). Intranasally infected mice develop an acute respiratory illness that is rapidly resolved by CD8+ T cell-mediated clearance of replicating virus in the lung (3,4). Latent infection, which persists for the life of the animal, is subsequently established in B cells, epithelial cells and macrophages (59). Associated with the establishment of latency is an EBV-like infectious mononucleosis (IM) phase, characterized by splenomegaly and the presence of activated CD8+ T cells in the peripheral blood (6,10). A feature of MHV-68-associated IM that distinguishes it from EBV-associated IM is the pronounced, selective expansion of CD8+ T cells utilizing Vß4 chains in their
ßTCR (10). An extensive analysis of ~18 strains of MHV-68-infected mice confirmed that the Vß4 expansion is not MHC restricted, although the extent of Vß4 expansion varies greatly among strains (10; Hardy et al., submitted for publication).
The
ßTCR is composed of highly polymorphic
and ß chains encoded by rearranging V, (D), J and C gene segments. The high degree of sequence polymorphism in the complementarity-determining region (CDR)-3 is conferred by the recombination of these gene segments. Junctional region diversity is further increased by the random insertion of nucleotides, resulting in variation in size of the CDR3ß (reviewed in 11,12). The
ßTCR recognizes fragments of antigen (peptide) bound between the
helices of MHC molecules, with the CDR3ß region providing important contacts with the bound peptide (13,14). A hallmark of T cell clones amplified during antigen-specific immune responses is the conservation of CDR3ß amino acid sequence or length (1518). In contrast, superantigen (sAg) binds directly to the outside of the MHC class II molecule and to the external face of the TCR Vß element, and T cell recognition of sAg is typically independent of CDR3 sequence (1922).
The Vß4-biased reactivity that is MHC haplotype independent in MHV-68induced IM is consistent with sAg reactivity (19,20), although we have previously reported the apparent absence of a requirement for MHC class II presentation, arguing against conventional sAg reactivity (23). Some reports have implicated sAg in the Vß-specific expansions seen during EBV-associated IM (24,25). However, in other reports, analysis of numerous patients with acute IM did not support the existence of a sAg-driven response. For example, it was shown that CD8+ T cells in the peripheral blood during EBV-associated IM are oligoclonal or monoclonal, and that a significant proportion of these activated CD8+ cells represent cytotoxic T lymphocytes (CTL) reactive to both lytic and latent EBV epitopes. Consistent with a role for viral antigen in driving the CD8+ expansions in IM, the CDR3ß showed clear evidence of selection of conserved amino acid motifs (26,27). There is relatively little information on the epitopes that drive T cell responses to MHV-68. However, the expanded Vß4+CD8+ T cells do not appear to be reactive to several CD8+ T cell epitopes defined in H-2b mice (28,29).
The nature of the Vß4 stimulatory ligand in MHV-68 infection remains elusive. Stimulatory activity in the spleens of infected mice correlates with the peak of viral latency, raising the possibility that a latent viral antigen is driving the Vß4+CD8+ T cells. However, the stimulation of Vß4+CD8+ T cells is apparently MHC independent (23). For example, analysis of a panel of Vß4+ T cell hybridomas generated from MHV-68-infected mice during the IM stage of infection showed reactivity to latently infected spleen cells from mouse strains that are deficient in MHC class I and II molecules. In addition, anti-MHC class I and II antibodies failed to block hybridoma recognition of latently infected spleen cells. Finally, expansion of Vß4+CD8+ T cells was observed in TAP1 and ß2-microglobulin knockout mice, that are deficient in MHC class I molecules, as well as in CD1 knockout mice.
In order to shed additional light on the nature of this unusual T cell reactivity, we sought to characterize the repertoire of responding Vß4+ T cells. In the current studies, we analyzed CDR3 size and TCR ß clonotype distribution of Vß4 chains amplified during the IM stage of MHV-68 infection using a PCR- and sequencing-based strategy (reviewed in 16). Our results clearly demonstrate the presence of multiple clonal and oligoclonal expansions within the Vß4+CD8+ population. These expansions dramatically distort the Vß4+ T cell repertoire and suggest an important role for the CDR3 region of the
ß TCR in the Vß4+CD8+ expansion associated with the IM-like phase of MHV-68 infection.
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Methods
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Mice
Female C57BL/6 mice, 812 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed under specific pathogen-free conditions until MHV-68 infection and in BL3 containment after infection. All animal procedures in these experiments were approved by the Institutional Animal Care and Use Committee at St Jude Children's Research Hospital, Memphis, TN.
Virus stocks and plaque assay
The original stock of MHV-68 (clone G2.4) was obtained from Professor A. A. Nash, (University of Edinburgh, Edinburgh, UK). Virus was grown in owl monkey kidney cells (ATCC, Rockville, MD; 1566 CRL) and titered by plaque assay on NIH 3T3 cells (ATCC; CRL 1568), as previously described (4).
Infection of mice and tissue sampling
Mice were anesthetized via i.p. injection of ~300 µl avertin (2% 2,2,2-tribromoethanol and 2% t-amyl-alcohol), then infected intranasally with 400 p.f.u. of MHV-68 in 30 µl HBSS. Mice to be sacrificed were given 400 µl avertin i.p. and ~100 U heparin sodium (1000 USP U/ml; Fujisawa, Healthcare Inc, Deerfield, IL). Blood (7501000 µl per mouse) was collected from an incision in the left ventricle, and transferred to a 15 ml tube containing 10 ml of HBSS and heparin (0.6 USP U/ml; Sigma, St Louis, MO; cat. no. 210-6) and stored on ice until processing. Blood was collected from MHV-68-infected mice 1 month post-infection.
V
repertoire analysis
The TCR V
repertoire was assessed using mAb to V
2 (B20.1), V
3.2 (RR3-16), V
8 (B21.14) or V
11.1, 11.2 (RR8-1). Antibodies were either biotinylated and used in association with streptavidinphycoerythrin (PE) (Biosource, Camarillo, CA) or PE conjugated. Anti-V
antibodies were either generated from concentrated culture supernatant or purchased (PharMingen, San Diego, CA), and used in a three-color staining protocol with Vß4FITC (KT4) (PharMingen) and CD8TriColor (CT-CD8a; Caltag, Burlingame, CA). Cells were stained in 96-well round-bottom plates, and 100,000 events were acquired on a FACScan and analyzed using CellQuest software (Becton Dickinson, San Jose, CA).
In vivo CD4 depletion of mice
GK1.5 anti-CD4 antibody was grown in a mAb production module (Techne, Princeton, NJ). Mice were injected i.p. with 0.5 ml of GK1.5 antibody for 7 days every second day. Depletion efficacy was monitored by staining peripheral blood lymphocytes (PBL) with CD4FITC clone RM4-4, which is not blocked by GK1.5, Vß4PE (PharMingen) and CD8TriColor (Caltag). Depletion of CD4 cells was ~95%. The percentage of CD8+ T cells in the peripheral blood of CD4-depleted mice was 5.9 ± 0.5% for naive mice and 17.7 ± 4.5% for MHV-68-infected mice.
Extraction of RNA and preparation of cDNA
RNA was extracted from CD4-depleted PBL ( >106 lymphocytes per mouse) using the Qiagen RNeasy kit and eluted in a volume of 40 µl (cat. no. 74104). Then 38 µl of RNA was denatured at 95°C for 5 min and the following sequentially added: 2 µl RNasin (Promega; cat. no. N2511), 16 µl 5xfirst-strand buffer (Gibco/BRL, Gaithersburg, MD), 8 µl dNTP (10 mM), 4 µl random primers (Gibco/BRL; cat. no. 48190-011), 8 µl 0.1 M DTT and 4 µl Superscript II RT (Gibco/BRL; no. 18064-014). The mixture was incubated at room temperature for 5 min, followed by 42°C for 1 h and finally at 95°C for 5 min, and stored at 20°C.
PCR reactions and CDR3 size analysis
PCR reactions were performed in 50 µl reaction volumes using a Perkin-Elmer 480 thermal cycler (Perkin-Elmer, Foster City, CA). First round PCR was performed by adding 1 µl of cDNA to the following mixture: 5 µl 10xPCR buffer (Perkin-Elmer), 3 µl MgCl2 (25 mM; Perkin-Elmer), 4 µl dNTP (10 mM each; Perkin-Elmer), 5 µl 5' primer and 5 µl 3' primer (10 µM each), 0.25 µl Taq polymerase (Perkin-Elmer), and 28 µl milliQ H2O. PCR reactions were carefully optimized and run to saturation. PCR conditions were as follows: 2 min at 95°C, followed by 35 cycles of 95°C for 1 min, 65°C for 1 min and 72°C for 50 s. A final extension at 72°C for 7 min was performed. PCR products were visualized following electrophoresis on 2% agarose gels (NuSieve 3:1 agarose; FMC BioProducts, Rockland, ME) with ethidium bromide staining. Unlabeled Vß4, Cß and Jß, and 3' Fam-labeled Cß and Jß primers (30) were synthesized (Center for Biotechnology, St Jude Children's Research Hospital). PCR run-off reactions were performed by adding 1 µl first-round PCR product to the following: 1 µl 10xPCR buffer, 0.6 µl MgCl2 (25 mM), 0.8 µl dNTP (10 mM each), 1 µl 3' Fam-labeled primer (10 µM), 0.25 µl Taq polymerase and milliQ H2O added to 10 µl. Run-off conditions were as follows: 2 min at 95°C, followed by 59 cycles of 95°C for 1 min, 65°C for 1 min and 72°C for 50 s. A final extension at 72°C for 7 min was performed.
Fam-labeled run-off products were denatured at 95°C for 2 min. Then 2 µl of run-off product was mixed with 2 µl loading buffer and 1 µl size standards (Genescan 1000 ROX; Applied Biosystems, Brisbane, Australia) and separated on a 6% polyacrylamide gel using an Applied Biosystems 373A DNA sequencer. Data were analyzed using Genescan analysis 2.1 Software (Applied Biosystems) which records the size and fluorescence intensity of each peak. The area of each individual peak was expressed as a percentage of total peak area. Only profiles with fluorescence intensity >700 U were analyzed. Major expansions were those which were at least 50% greater than the highest corresponding value in naive mice. CDR3 sizes were calculated from codons 95106, inclusive (11).
Cloning of PCR products for VDJ junctional region sequencing
Excess primers and nucleotides were removed from Vß4Cß PCR products using the Qiagen PCR purification kit (cat. no. 28104), according to the manufacturer's instructions. An aliquot of 1 µl of this cleaned product was used as a template for subsequent Vß4Jß reactions using the above PCR conditions. Vß4Jß PCR product quality was checked by gel electrophoresis (as above) and 1.5 µl of the reaction product cloned using the TOPO TA cloning kit and pCR®II-TOPO vector (Invitrogen; cat. no. K460001). White insert-containing colonies were picked and grown for 1618 h in 2.5 ml LB medium at 37°C on an orbital shaker. DNA purification was performed using the Qiagen Miniprep kit (cat. no. 27106). Samples of 10 µl of DNA were sequenced on an Applied Biosystems 377 sequencer (Center for Biotechnology, St Jude Children's Research Hospital) using M13-21 and reverse primers, and TaqFS dye terminator chemistry. Sequences were analyzed using GCG software (Wisconsin Package).
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Results
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Major CDR3-restricted distortions in the Vß4+CD8+ T cell population
Our previous studies of MHV-68 infection have shown that expansion of Vß4+CD8+ T cells is seen in mice of diverse MHC haplotypes (10; Hardy et al., submitted for publication). At present it is unclear what drives this dramatic T cell expansion and whether it is composed of clonally or polyclonally restricted populations. To determine the clonal composition of the expanded cells, Vß4 region repertoire diversity was characterized using a PCR- and sequencing-based protocol that determines CDR3 length and clonotype distribution (30). This technique provides a global overview of diversity within given Vß families and also permits focused analysis of individual Jß subfamilies. The presence of clonal or oligoclonal expansions is demonstrated by distortion of an otherwise Gaussian distribution of peak sizes (16).
In order to avoid analysis of the CD4+ T cell CDR3 region, C57BL/6 mice were depleted of CD4 cells by in vivo injection of GK1.5 antibody. Previous studies from our laboratory demonstrated that Vß4+CD8+ T cells had reduced viability ex vivo (unpublished observations), and for this reason we avoided potentially deleterious manipulations such as in vitro depletion and flow cytometric sorting. Additionally, we have shown that depletion of CD4+ cells during the first week post-infection prevents expansion of Vß4+CD8+ T cells (31). However, depletion subsequent to 14 days post-infection has no effect on the magnitude of Vß4+ expansion (31). Mice in the current study were depleted from 22 days post-infection.
Initially, CDR3 size analysis of the entire Vß4 family using Fam-labeled Cß in the run-off reaction was performed on four naive mice and six MHV-68-infected mice. Peripheral blood cDNA from CD4-depleted naive mice yielded Gaussian CDR3 size distributions (Fig. 1
shows the data for two representative naive mice) indicative of a diverse or polyclonal repertoire, as expected. In contrast, there was clear evidence of repertoire distortion among Vß4 chains in five out of six infected mice (Fig. 1
shows the data for two representative mice). Infected mouse #1 showed a prominent expansion of CDR3 14 amino acids in length (10% of the repertoire), whilst CDR3 of this size were not seen in either naive mouse. Infected mouse #2 showed a prominent expansion of CDR3 11 amino acids in length (29%), with the corresponding values for the naive mice being 14 and 17% respectively. Flow cytometric analysis of the two naive mice indicated that ~6% of CD8+ cells expressed Vß4+
ßTCR, whilst the corresponding values for MHV-68-infected mice (infected #1 and infected #2) were 31 and 42%, respectively.

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Fig. 1. Vß4Cß CDR3 size distribution in CD8+ T cells from naive and MHV-68-infected mice. cDNA extracted from CD4-depleted peripheral blood of naive mice and mice 1 month post-MHV-68 infection was subjected to PCR amplification using Vß4 and Cß primers, followed by a run-off reaction with Fam-labeled Cß primer. Products were separated on a sequencing gel and size distribution analyzed using appropriate software. Relative fluorescence intensity, plotted on the y-axis, was always >700 U. Expansions at CDR3 of 14 or 11 amino acids were detected in the infected mice as indicated (asterisk).
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Having established that the Vß4Cß repertoire is non-uniformly distorted in MHV-68-infected mice, we determined the extent of Jß gene involvement by CDR3 size analysis using 3' primers to each of the 12 mouse Jß genes. The fact that Vß4 expansion has been consistently seen in all C57BL/6 mice examined to date (data not shown) justified focussing the detailed analysis on two individual naive and two individual infected animals. The CDR3 size distribution for most Jß elements in naive mice was overall normally distributed (polyclonal) (Fig. 2
). In contrast, the CDR3 size distribution in each of the infected mice was markedly skewed across most Jß repertoire profiles (Fig. 2
). Infected mouse #1 showed prominent expansions in each of the Jß subfamilies, whilst infected mouse #2 showed prominent expansions in all but the Jß2.5 subfamily. In every case an expansion was considered significant if the percentage of the repertoire at a particular CDR3 size was at least 50% greater than the corresponding value in either naive mousethe expansions in infected mice were frequently
200% of the values in naive mice. It is noteworthy that the expansion in the Jß2.3 subfamily at CDR3 of 14 amino acids in infected mouse #1, which represented 57% of the repertoire, was also detected in the Vß4Cß profile (Fig. 1
). In contrast, CDR3 of 14 amino acids were undetectable in the Vß4Cß and Vß4Jß2.3 analysis of either naive mouse. Overall, the MHV-68-induced Vß4+CD8+ expansion appears to be unusual in that it consists of multiple Vß4Jß selective expansions that impact dramatically on the available diversity within the circulating repertoire.
VDJ junctional region sequencing demonstrates clonal and oligoclonal expansions
CDR3 size-restricted expansions are strong evidence of selective clonal or oligoclonal expansions (1618). To determine the distribution of individual clonotypes within the Vß4+CD8+ expansions in infected C57BL/6 mice we performed VDJ junctional region sequencing. PCR products from two Jß subfamilies which showed expansions at identical CDR3 sizes in the two infected mice, Jß1.6 and Jß2.2, were sequenced to determine whether similar or identical TCR sequences had been selected. Sequences obtained from cloned Vß4Jß1.6 PCR products in infected mouse #1 were predominantly 10 amino acids in length (87%), with one major clonal expansion (with a junctional amino acids sequence of QDAGN) which represented 70% of total sequences (Fig. 3
). Similarly, the majority of Vß4Jß1.6 sequences in infected mouse #2 were 10 amino acids in length (75%), being comprised of one dominant clone (QEWGL, 38%; Fig. 3
).

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Fig. 3. Vß4Jß1.6 junctional region sequences in MHV-68-infected mice. cDNA extracted from CD4-depleted peripheral blood of two individual mice 1 month post-infection was subjected to PCR using Vß4 and Cß primers. A second PCR reaction was performed with Vß4 and Jß1.6 primers using 1.5 µl of the above PCR product as template. The PCR reactions were performed completely independently of those for CDR3 size analysis. The PCR products were cloned and DNA from insert-containing colonies sequenced. Two nucleotide sequences encode for the NDN sequences QDAGN and QEWGL in mouse #1 and #2 respectively. The differences in the nucleotide sequences of the relevant codons are in bold and underlined. The frequency of prominent expansions is in bold. CDR3 size was calculated from codons 95106 inclusive.
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Sequence data obtained from cloned Vß4Jß2.2 PCR products in infected mouse #1 demonstrated that 96% of sequences had CDR3 sizes of 11 amino acids, with two clones detected which represented 57 and 25% of total sequences (QDWDA and QDLGWD respectively; Fig. 4
). Whilst the majority of Vß4Jß2.2 sequences in infected mouse #2 were also of 11 amino acids (80%), as predicted by CDR3 size analysis, three obvious clonal expansions (QDWDA, QDWGA and QDWGAG) were detected which accounted for 28, 16 and 20% of total sequences respectively. The glutamine (Q) in these CDR3 is presumably derived from the Vß4 germline sequence (32), although amino acids in this position can be non-germline encoded. Nevertheless, there appears to be selective pressure to maintain the DWXA motif derived in part from the Dß2.1 germline sequence (33). An additional expansion at CDR3 of 12 amino acids (QEEGGGA; 16% of sequences) was also seen (Fig. 4
). Thus, although both infected mice had Vß4Jß1.6 expansions with a CDR3 of 10 amino acids, these were composed of clones with distinct sequences. Although several of the 11 amino acid Vß4Jß2.2 expansions in the two infected mice were unique, one clone with identical nucleotide sequence (QDWDA; Fig. 4
) was seen in both mice.

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Fig. 4. Vß4Jß2.2 junctional region sequences in MHV-68-infected mice. The PCR reactions and sequencing were performed as described in the legend to Fig. 3 . In infected mouse #1 two nucleotide sequences encode for the NDN sequence QDWGA. The differences in the nucleotide sequences of the relevant codons are in bold and underlined. The NDN sequences QDWDA (indicated by double asterisks) had identical nucleic acid sequences in both mice. The frequency of prominent expansions is in bold. CDR3 size was calculated from codons 95106 inclusive.
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It is important to note that CDR3 size analysis and VDJ sequencing gave similar profiles for CDR3 length distribution (Table 1
). For example, in the Vß4Jß2.2 analysis of infected mouse #2, CDR3 size analysis showed that CDR3 of 11 and 12 amino acids represented 62 and 26% of the repertoire respectively. This was consistent with sequencing data, which showed that CDR3 of 11 and 12 amino acids represented 80 and 16% of the repertoire respectively (Table 1
). The slight difference in absolute values from either method may reflect the statistical error imposed by sequencing a limited number of clones. Significantly, the PCR reactions for the two analyses were performed completely independently, thereby providing confirmation of the reproducibility and validity of the two PCR-dependent techniques.
Thus, the sequencing of Jß1.6 and Jß2.2 chains showed evidence for oligoclonal and clonal expansions, some of which were shared between the two individual mice sequenced. These data are consistent with earlier random sequencing of Vß4Cß VDJ junctional region sequences from four individual MHV-68-infected mice, which also showed evidence for unique and `public' expansions (data not shown).
VDJ junctional region sequences in naive mice
Although the CDR3 size analysis results from the naive mice showed largely Gaussian profiles for the individual Jß subfamilies, VDJ junctional region sequencing was performed in naive mouse #2 to determine whether individual Jß subfamilies were composed of distinct populations of cells. VDJ junctional region sequencing from the Jß1.6 subfamily showed that there was a diverse distribution of CDR3 sizes (Fig. 5
and Table 1
), although several repeat sequences of a CDR3 size of 12 amino acids (QDRVN) were found. VDJ junctional region sequences from the Jß2.2 subfamily showed 10 unique sequences and a CDR3 size distribution which broadly mirrored that obtained using CDR3 size analysis (Fig. 6
and Table 1
).

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Fig. 5. Vß4Jß1.6 junctional region sequences in a naive mouse. The PCR reactions and sequencing were performed as described in the legend to Fig. 3 , using cDNA for naive mouse #2. Two nucleotide sequences encode for the ND-N sequence NKH. The differences in the nucleotide sequences of the relevant codons are in bold and underlined. CDR3 size was calculated from codons 95106 inclusive.
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Fig. 6. Vß4Jß2.2 junctional region sequences in a naive mouse. The PCR reactions and sequencing were performed as described in the legend to Fig. 3 . CDR3 size was calculated from codons 95106 inclusive.
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V
usage by Vß4+CD8+ T cells
To further characterize receptor diversity, we analyzed V
usage by Vß4+CD8+ cells (Table 2
). The data show that V
8 was consistently paired with Vß4 at higher frequency in infected compared to naive mice, in both spleen (22.1 ± 3.4 versus 8.9 ± 1.1 % respectively) and blood (20.8 ± 6.6 versus 6.9 ± 1.1 % respectively). However, this was not unique to Vß4 chains, as there was also an increase in V
8 usage among all CD8+ T cells from infected mice. In one mouse expansion of V
3.2+
chains was observed exclusively in the Vß4+ subset of CD8+ T cells in both spleen and blood, whilst this change was not seen in the other three mice. There was little change in the frequency of V
2 usage, and a small compensatory reduction in relative usage of V
11 in both splenic and peripheral blood Vß4+CD8+ T cells as a consequence of MHV-68 infection. These results indicate that there is diversity in the V
repertoire of Vß4+CD8+ T cells in MHV-68-infected mice.
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Discussion
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Infection of mice with MHV-68 causes a dramatic expansion of peripheral blood CD8+ T cells which, in some aspects, mirrors EBV-induced IM. However, unlike EBV, there is a strong bias among the peripheral blood T cells during the IM phase toward those that utilize Vß4 chains in their
ßTCR, resulting in an increased percentage of Vß4+ T cells among the CD8+ T cells. The Vß4+CD8+ T cell expansion occurs in virtually all mouse strains examined, although to varying extents, and the effect has been shown to be independent of MHC haplotype (10; Hardy et al., submitted for publication). The nature of the stimulatory ligand remains elusive, but it does not appear to be dependent on MHC class I or II molecules nor on the non-classical MHC class I molecule CD1 (23). We investigated CDR3 size restriction and VDJ junctional region sequence diversity of Vß4+CD8+ T cells solely in C57BL/6 mice since this strain shows the most pronounced expansion and has been used extensively to study the biology of MHV-68 infection (4,23,34). Our results clearly indicate the presence of multiple clonal and oligoclonal expansions within the Vß4+CD8+ T cell subset amplified during the IM phase of MHV-68 infection.
T cells specific for lytic and latent epitopes have been identified in EBV-infected patients (3537) and large oligoclonal expansions are seen within the activated CD8+ set during acute IM (26,27). Additionally, clonal expansions to a latent EBV epitope were found in healthy EBV-seropositive individuals (38). Despite these findings in EBV, it appears that at least the Vß4+ component of the activated CD8+ T cells in the peripheral blood during the IM phase of MHV-68 infection is not specific for lytic or latent epitopes. Several acute phase MHV-68 viral epitopes in H-2b mice (28,29,39) and a latent CTL epitope in H-2d mice have been identified (40). In contrast to the situation for EBV (27), T cells specific for lytic epitopes are relatively infrequent, generally 111% of the blood CD8+ set (28). In addition, with the emergence of Vß4+CD8+ T cells in the peripheral blood at ~20 days post-infection, the frequency of CD8+ T cells specific for the lytic epitopes drops (28). Analysis of a panel of Vß4+CD8+ hybridomas generated from MHV-68-infected mice during the IM phase and reactive to latently infected spleen cells (23) failed to identify reactivity to the lytic or latent epitopes (unpublished data).
A role for sAg in herpesviruses has previously been suggested (24,25,41). In addition, a product of the herpesvirus saimiri ORF14, that is homologous to the MMTV sAg, Mtv7, encodes a protein that binds to MHC class II molecules and stimulates T cells. However, this activity differs from a conventional sAg because there is no Vß bias to the polyclonal T cell activation (42). This product has recently been shown to play an essential role in T cell transformation by the virus, and for high-level persistent infection (43).
We previously postulated that the striking Vß4 bias of the CD8+ T cell expansion may be due to the presence of a sAg (10). Hallmarks of sAg responses are dependence on class II MHC expression, lack of MHC restriction, stimulation of specific Vß-bearing T cells and lack of antigen-processing requirement (19,20). The presence of oligoclonal expansions described here are unusual for known sAg. Contact between the TCR and bacterial or viral sAg is largely dependent upon interactions with CDR1, CDR2 and the hypervariable region 4 (HV4) loop of the TCR ß chain (1922), distinct from the CDR3 region which is predominantly responsible for the fine antigen specificity of the T cell (13). Consistent with this, CDR3 size analysis of sAg-reactive T cells has failed to show evidence for CDR3 involvement in two reported studies (44,45). Nevertheless, the TCR
chain and CDR3ß sequence may stabilize TCRsAg interactions which are suboptimal or involve unusual sAg (4649). This is illustrated by the finding that T cell recognition of the sAg produced by Mycoplasma arthritidis mitogen (MAM) is dependent upon two amino acids located within the Vß17 CDR3 region (50). Thus, although the CDR3-size restricted oligocolonal populations of T cells and the putative `public' expansion (QDWDA) seen in Vß4Jß2.2 sequences are characteristic of antigen-driven expansions, it remains formally possible that the expansion is mediated by an unusual sAg, similar to MAM. Although sAg have been reported to modulate CD8+ T cell responses (51,52), the possibility that a putative MHV-68-associated sAg reactivates Vß4+CD8+ T cells expanded during the lytic phase seems remote as these cells are not reactive to known lytic epitopes (28).
Numerous studies have demonstrated TCR Vß-specific expansions in response to virus infection in mice (5356). However, whilst all of the above Vß-specific expansions are driven by known peptideMHC combinations, they differ from the Vß4 expansion in MHV-68-infected mice which is MHC independent (10,23). Lin and Welsh examined the Vß8.1+ T cells responding to lymphocytic choriomeningitis infection, and demonstrated a surprisingly diverse repertoire which was unique to each mouse examined (57). A diverse Vß17+ T cell repertoire is also seen during the response to influenza A matrix peptide M15866 in the human (58). Others have shown highly restricted repertoires in response to a number of viral and non-viral peptideMHC antigens (17,18,55,59). Thus, whilst CDR3ß amino acids sequence restriction or diversity does not in itself indicate that the immune response is driven by peptideMHC, the presence of dominant clonal expansions is generally seen to support this conclusion.
The question of which T cells from the acute repertoire are selected into the memory repertoire remains controversial (17,59). In the case of lymphocytic choriomeningitis infection it appears that the memory repertoire evolves from a representative sampling of the acute T cell repertoire (56,57), consistent with findings in other systems (17). However, there is evidence that the T cell repertoire during recall responses, at least in the case of Listeria monocytogenes infection, may be more `focussed' (60). It would be of interest to perform longitudinal studies of individual mice to determine whether the Vß4+CD8+ T cell repertoire evolves or remains stable over time.
It would be anticipated that naive mice should have a Gaussian CDR3 size distribution. However, VDJ junctional region sequencing of the Jß1.6 subfamily showed evidence of a small clonal expansion, although the Vß4Jß1.6 CDR3 size distribution for this mouse was not markedly skewed. Biases in Jß usage are seen in the developing and mature T cell repertoire, resulting in under-utilization of certain Jß elements, including Jß1.6 (47,61,62). This bias may make the sample size of the Jß1.6 pool statistically unrepresentative compared to more abundantly expressed groups such as Jß2.2. Thus, even small clones of cells may be sufficient to distort the repertoire, thereby giving rise to repeat sequences in naive mice. In naive C57BL/6 mice, memory/activated (CD62Llo) CD8+ T cells are somewhat enriched in the blood compared to the spleen (data not shown). This raises the possibility that clonally expanded cells are relatively frequent in the blood and may account for some skewing of the `naive' repertoire.
The oligoclonality of the expanded Vß4+CD8+ T cells is consistent with the idea that a small subset of MHV-68-reactive Vß4+ precursors exists in the naive repertoire and are preferentially stimulated to proliferate. This hypothesis is supported by the finding that a low frequency of Vß4+ T cells respond to MHV-68-infected splenocytes in vitro and is consistent with the finding that a low frequency of random Vß4+CD8+ T cells hybridomas (~ 3%) can be stimulated by MHV-68-infected spleen cells (unpublished observations). This possibility is concordant with the ~1 week lag between detection of the stimulatory activity in the spleen and observed elevated levels of Vß4+CD8+ T cells in the peripheral blood (10,23; Hardy et al., submitted for publication). Nevertheless, the stimulus driving proliferation is strong, since >3050% of CD8+ T cells are Vß4+ 34 weeks post-infection. This idea is reinforced by the finding that large expansions of Vß4+CD8+ cells are found in TAP1 and ß2-microglobulin knockout mice which have low numbers and a restricted repertoire of CD8+ T cells (23,28,63,64).
In summary, the Vß4+CD8+ T cells that dominate the activated T cells in the peripheral blood during MHV-68-induced IM represent preferentially amplified CDR3 size-restricted oligoclonal populations, a finding typical of peptideMHC-driven T cell proliferation, but also consistent with unusual sAg, such as MAM. Taken together with previous studies suggesting that the reactivity is apparently independent of MHC class I and class II molecules, as well as ß2-microglobulin or TAP1 expression (23), the data suggest that the ligand driving Vß4 expansion is unconventional and neither fits into the sAg or antigen-specific TCR recognition paradigms.
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Acknowledgments
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The authors thank Drs Edward Usherwood and In-Jeong Kim for critical evaluation of the manuscript, Carole Rohl for assistance with the preparation of the manuscript, and Scottie Adams (Molecular Biology Core Facility, Trudeau Institute, Saranac Lake, NY) and Phuong Nguyen (Department of Immunology, St Jude Children's Research Hospital) for technical assistance. This work was supported by NIH grant AI42927 (MAB), P30 CA21765 (CORE grant), the American Lebanese Syrian Associated Charities (ALSAC), and the Australian National Health and Medical Research Council.
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Abbreviations
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CDR complementary-determining region |
CTL cytotoxic T lymphocyte |
EBV EpsteinBarr virus |
IM infectious mononucleosis |
MAM Mycoplasma arthritidis mitogen |
MHV-68 murine -herpesvirus-68 |
PBL peripheral blood lymphocyte |
SAg superantigen |
 |
Notes
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4 Present address: CRC for Asthma, Department of Pathology and Immunology, Monash Medical School, Prahran, Victoria 3118, Australia 
5 Present address: Trudeau Institute, Saranac Lake, NY 12983, USA 
Transmitting editor: E. Simpson
Received 7 January 2000,
accepted 2 May 2000.
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