In vivo selection of a TCR Vß repertoire directed against an immunodominant influenza virus CTL epitope

Weimin Zhong and Ellis L. Reinherz

Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, Boston, MA, USA

Correspondence to: W. Zhong; E-mail: weimin_zhong{at}dfci.harvard.edu

Correspondence to: E. Reinherz; E-mail: ellis_reinherz{at}dfci.harvard.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Little is known about the mechanisms governing TCR repertoire selection in response to foreign antigens. Here, we evaluate the molecular features of the murine C57BL/6 (B6) TCR Vß repertoire directed at the NP366–374 immunodominant epitope of the influenza virus nucleoprotein. Common or ‘public’ ß chains are shared among individuals following either primary or secondary infection. Importantly, repertoire diversity decreases substantially after a second viral exposure due to enrichment of TCRs sharing Vß CDR3 loops of identical length and highly related amino acid sequences. TCRs from these secondary T cell populations possess greater overall avidity for the NP366–374/Db complex compared to those from the primary repertoire. Thus, expansion of CD8+ T cells expressing a favored germline Vß gene segment in the primary response and further selection for CDR3ß loops during the secondary response, contribute to optimization of immune recognition against certain viral epitopes.

Keywords: influenza virus, repertoire, T cell receptor, vaccination


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity plays an important role in protection against viral infections (1). Virus-specific precursors proliferate and differentiate into functional effector cells after initial viral challenge. Following clearance of virally infected target cells, most of the primary CTL effectors undergo activation-induced cell death (AICD). Only a few of the effectors survive the AICD and become memory T cells. Memory T cells are capable of responding to a secondary viral challenge with accelerated kinetics and increased magnitude, leading to a more rapid viral clearance (2). One important aspect in developing T cell immunity-based vaccination strategies against viral infections is to understand and optimize these processes, of which the central recognition elements are highly diverse and clonally distributed {alpha}ß T cell receptors (TCRs).

TCRs are expressed primarily as {alpha}ß heterodimeric membrane-bound proteins and define the diversity, clonality and specificity of the T cell repertoire. To obtain sufficient diversity for recognition of a wide variety of foreign antigens, TCRs are generated via somatic recombination and imprecise joining of different germline variable (V), diversity (D) and joining (J) gene segments within the TCR ß-locus and V and J segments within the TCR {alpha}-locus. Random addition of template independent nucleotides at the V(D)J junctions during this process and pairing of numerous different {alpha} and ß chains further contribute to the diversity of TCR repertoire [reviewed in (3)]. The complementarity-determining region 1 (CDR1) and CDR2 loops of TCRs are encoded within the germline V gene segments and are less variable in amino acid sequence than the highly diverse CDR3 loops resulting from rearrangement of V(D)J segments. Recently solved crystal structures of TCR/peptide/MHC complexes have revealed that the CDR3s of both V{alpha} and Vß domains lie over a central position of the peptide/MHC complex, thus interacting predominantly with bound antigenic peptides [reviewed in (4,5)]. Functional analysis of the interaction between TCRs and the peptide/MHC complexes also support the idea that the sequence features and the length of CDR3 loops are two major determinants in recognition of peptides presented by MHC molecules (6).

A number of early studies have attempted to correlate TCR V gene usage with the recognition of a specific viral peptide/MHC complex. From these studies, it appears that TCR repertoires selected in response to viral infections are either extremely diverse (7,8) or highly restricted (9,10). However, most of these experiments involved the analyses of limited numbers of in vitro expanded T cell hybridomas and/or CTL clones, a process which may not accurately reflect the antigen-specific TCR repertoires in vivo since it is prone to experimental biases. Moreover, only primary TCR repertoires were analyzed in these studies. Therefore, little is known about the size, complexity and stability of TCR repertoires in vivo or attendant changes which occur during transition from primary to memory responses.

Recently, these issues have been re-addressed using ex vivo materials. Analysis of MHC class II-restricted CD4+ T cell response to pigeon cytochrome c (PCC) in mice showed that the TCR repertoire narrowed upon repeated exposure to the PCC antigen (11). A similar phenomenon was inferred from studying TCR Vß usage in an MHC class I-restricted CD8+ T cell response to an immunodominant CTL epitope of Listeria monocytogenes (12). No reduction in TCR diversity has been previously observed in any infectious models after a secondary antigen exposure (1315). However, the nature and sequence diversity of TCR CDR3 loops arising after a natural pathogen infection have seldom been analyzed ex vivo (16).

In the present study, we analyzed the clonal compositions and selection processes of the TCR Vß repertoire during a primary and secondary response to an MHC class I-restricted immunodominant CTL epitope of influenza A virus in B6 mice, NP366–374 (17,18). We found that the NP366–374-specific immune repertoire predominantly selected the Vß8.3 gene segment in vivo. Dominance of Vß8.3 usage was stable during primary, resting memory and secondary response to the virus. To dissect the molecular features of CDR3ß regions associated with recognition of the NP366–374/Db complex, NP366–374-specific bulk populations were first sorted to high purity directly ex vivo from the virus infected animals. The sorted materials were then used for subsequent analyses by spectratyping (19) and CDR3 junctional sequencing. This strategy allows us to precisely track any subtle changes of CDR3 molecular structures within the NP366–374-specific immune repertoire during the virus infection. Our results reveal that although substantial diversity exists during the primary response, certain TCR ß subunits are common or ‘public’ (20) for all individuals tested. Most importantly, we found a considerable increase in frequency of these public TCRs during the memory response, accompanying an increase in overall TCR affinity for the NP366–374 epitope.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Oligonucleotides and fluorescent dye labeling
Unlabeled and fluorescent dye 6-Fam-labeled oligonucleotides were synthesized at Intergrated DNA Technology, Inc. using an Applied Biosystems DNA synthesizer. Dye-labeled oligonucleotides were purified by reverse-phase chromatography to remove the unlabeled oligonucleotides. Primer sequences for mouse Vß8.3, Jß and Cß gene segments were described by Pannetier et al. (19).

Infection of mice with influenza virus
Female C57BL/6 mice were purchased from Taconic (Albany, NY) and housed under specific pathogen-free conditions at the animal core facility of the Dana-Farber Cancer Institute prior to infection with influenza virus at 6–10 weeks of age. Influenza viruses were grown, titered and stored as described previously (18). Primary infection was performed by inoculating 3000 EID50 of influenza A/PR8/8/34 (PR8, H1N1) viral particles intranasally under anesthesia. T cell memory status was considered established when mice had been infected with PR8 virus a minimum of 30 days previously. PR8 memory mice were re-challenged intranasally with 3000 EID50 of a serologically distinct influenza A/HK/x31 (x31, H3N2) virus to study the secondary CD8+ T cell response to the virus. Both viruses share six of the eight RNA segments in the genome of influenza A virus, including the nucleoprotein-encoding gene.

Tissue sampling and processing
Lung, draining mediastinal lymph nodes (MLN), peripheral blood (PB) and spleen were collected from B6 mice previously infected with the influenza A virus. Single cell suspensions were prepared from lung, MLN and spleen by passage through cell strainers. Lymphocytes from lung tissues were further enriched by 40–80% discontinue isotonic Percoll gradient centrifugation. Erythrocytes were removed from the cell preparations by treating with buffered ammonium chloride solution.

Flow cytometry
NP366–374/Db or PA224–233/Db tetrameric reagents were purchased from Beckman Coulter, Inc., Immunomics Operations, San Diego, CA. Cells were first stained with APC-conjugated NP366–374/Db or PA224–233/Db tetramer in combination with FITC-conjugated anti-mouse CD8{alpha} mAb (BD PharMingen) on ice for 45 min. For determination of TCRVß usage of the NP366–374-specific CD8+ T cells, a panel of 15 FITC-labeled anti-mouse Vß mAbs (PharMingen) was used in combination with Cy-Chrome-labeled anti-mouse CD8{alpha} mAb and PE-conjugated NP366–374/Db tetramer. After washing, the cells were acquired on a Becton Dickinson FACSCalibur flow cytometer and the data were analyzed using CellQuest software (Becton Dickinson Immunocytology System, San Jose, CA). The results were expressed as percentage of tetramer+CD8+ cells among total CD8+ T cells.

To sort antigen-specific T cells from the spleen of the virus-infected animals for spectratyping and CDR3ß junctional sequencing, single cell suspensions were stained with APC-conjugated NP366–374/Db tetramer together with PE-conjugated anti-mouse CD8{alpha} mAb and FITC-conjugated Vß8.3 mAb (BD PharMingen). NP366–374/Db+ Vß8.3+ CD8+ cells were then sorted on a MoFlo cell sorter (DAKO Cytomation, Fort Collins, CO). Vß8.3+ CD8+CD44low cells from spleens of naive B6 mice were sorted as controls. The purity of the sorted cells was typically >95% in this study.

The tetramer dissociation assay was performed according to the descriptions by Savage et al. with minor modifications (21). Briefly, lymphocyte-enriched single cell suspensions from the lungs of the virus-infected animals were first stained with NP366–374/Db tetramer and anti-mouse CD8{alpha} mAb as described above. The cells were then washed three times. One hundred micrograms of anti-mouse Db mAb (clone HB-27) was added to each well to block re-binding of dissociated tetramer to TCRs during the subsequent incubation process. Samples were collected at various time points after incubation, washed and fixed with 2% paraformaldehyde solution before analysis. All of the samples were analyzed by flow cytometer as described above. Normalized fluorescence intensity of NP366–374/Db-positive cells was used to calculate the slope and half-lives (t1/2) of NP366–374/Db tetramer dissociation.

RNA and cDNA preparation
Total RNA was extracted from ~10 000 sorted cells using the RNeasy Mini kit (Qiagen) and eluted in a volume of 35 µl. Total RNA was then treated with DNase I (Promega) to eliminate any trace amount of genomic DNA in the RNA preparations. Single-strand cDNA was synthesized using a first-strand cDNA synthesis kit (Roche) according the manufacturer's instruction. DNase I-treated RNA (10.2 µl) was used for each 20 µl of reaction.

Spectratyping
Spectratyping analysis of the CDR3 size distribution was modified according to the original description by Pannetier et al. (19). Briefly, 2 µl of cDNA preparations, corresponding to ~20 ng of RNA, was added to the following mixture in a total reaction volume of 50 µl: 5 µl of 10-fold concentrated Tris buffer (pH 8.2), 1 µl of 10 mM dNTP mix, 1.5 µl of 50 mM MgCl2, 2.5 µl of 10 pM 6-Fam-labeled 5'-Vß8.3 primer, 2.5 µl of 10 pM 3'-Cß primer or each of the 12 3'-Jß primers, and 0.25 µ1 (1 unit) of Ampli Taq DNA polymerase (Applied Biosciences) and 37.25 µl of MilliQ H2O. PCR was run as follows: 2 min at 95°C, followed by 36 cycles of 95°C for 1 min, 65°C for 1 min and 72°C for 50 s. A final extension step was performed at 72°C for 10 min. The PCR runs reached saturation under this condition as assessed by agarose gel electrophoresis of PCR aliquots taken at different cycles. PCR products (1.5 µl) were mixed with 4 µl of loading buffer containing GeneScan 500 ROX size standard (Applied Biosciences) and denatured at 95°C for 2 min. Mixtures (0.5–1 µl) were then loaded onto a 6% polyacrylamide gel and separated using an Applied Biosciences 373A DNA sequencer (Molecular Biology Core Facility, Dana-Farber Cancer Institute). The data were analyzed by GeneScan and/or Genotyper softwares (Applied Biosciences). CDR3ß length was calculated according to Rock et al. (22).

Cloning of PCR products for CDR3 junctional sequencing
Excess primers and oligonucleotides were removed from Vß8.3-Cß PCR products using the QIAquick PCR purification kit (Qiagen). One microliter of the purified PCR products was cloned into pCRII-TOPO vector using the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instruction. Single white colonies were picked and grown in 2 ml of LB medium containing kanamycin at 50 µg/ml at 37°C for 16–18 h on an orbital shaker. Plasmids were purified using the Qiaprep spin miniprep kit (Qiagen). Two micrograms of plasmid DNA was sequenced on an Applied Bioscience 377 DNA sequencer using forward and reverse M13 primers (Molecular Biology Core Facility, Dana-Farber Cancer Institute). Sequences were analyzed using DNAStar software (DNASTAR Inc.). Jß assignment was done according to Malissen et al. (23). The number of bacterial colonies with distinct nucleotide sequences and their deduced amino acid sequences of CDR3ß regions was used to measure the complexity of TCR Vß repertoire for the NP366–374 epitope.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Primary versus secondary CD8+ T cell responses to immunodominant CTL epitopes of influenza A virus
CD8+ T cell responses to MHC class I-restricted epitopes of influenza A virus in B6 mice have been extensively studied (17,18). It has been established that NP366–374 and PA224–233 are the two immunodominant CTL epitopes during primary infection. However, an altered hierarchy for the two epitopes during secondary infection has been documented (24). In order to decide which of the two epitopes was a more suitable target for the analysis of antigen-driven TCR repertoire selection during transition from primary effectors to memory effectors, we carefully re-examined this phenomenon using NP366–374/Db and PA224–233/Db tetrameric reagents. As expected, minimal tetramer-positive staining was observed on CD8+ T cells from lung, MLN and spleen of naive B6 mice (Fig. 1). Consistent with the previous studies (17,18,24), comparable staining profiles (11.3–14.5%) for both NP366–374 and PA224–233 epitopes were observed at the peak of a primary CD8+ T cell response to the A/PR8/8/34 (PR8) virus (Fig. 1). The frequency of PA224–233-specific CD8+ T cells in the major inflammatory site (lung), the secondary lymphoid organs (MLN and spleen) (Fig. 1) and in the circulation (PB) (data not shown) was equivalent or even higher than that of NP366–374-specific CD8+ T cells in these anatomic sites. When PR8 memory mice were re-challenged intranasally with the related A/HK/x31 virus, a massive expansion of NP366–374-specific CD8+ T cells was observed in all of the four tissues tested. NP366–374-specific CD8+ T cells comprised >70% in the lung and >20% in the spleen. In contrast, no obvious expansion of the PA224–233-specific CD8+ T cells was detected at the peak of the secondary infection (Fig. 1B). The frequencies of the PA224–233-specific CD8+ T cells were essentially comparable to those observed in a primary memory pool to the epitope (data not shown). Lacking experimental evidence that PA224–233-specific CD8+ T cells can undergo significant expansion corresponding to the magnitude of a typical memory response after secondary viral challenge, we chose the NP366–374 epitope for further experimentation in the present study.



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Fig. 1. Primary versus secondary CD8+ T cell responses of B6 mice to the two immunodominant CTL epitopes of influenza A virus. Primary infection was performed by inoculating B6 mice intranasally with influenza PR8 virus. Alternatively, mice infected with PR8 viral particles a minimum of 30 days previously were re-challenged intranasally with influenza x31 virus during a secondary infection. Naive uninfected B6 mice were used as controls. The primary (day 10 after the initial viral challenge) and secondary (day 7 after the secondary viral challenge) CD8+ T cell responses to NP366–374 and PA224–233 epitopes in various tissues were analyzed by flow cytometry using NP366–374/Db or PA224–233/Db tetrameric reagent (panels A and B, respectively). The numbers in the upper right quadrant of each dot plot represent the percentages of tetramer+CD8+ cells among the total CD8+ T cells examined.

 
Vß usage of NP366–374-specific CD8+ T cells
Deckhut et al. have previously shown that the Vß8.3 gene segment was dominantly used among a panel of NP366–374-specific T cell hybridomas generated after primary infection with influenza A virus (9). To obtain a global view of the Vß gene usage within the NP366–374-specific T cell responses in vivo, we analyzed the Vß repertoire of CD8+ T cells specific for this epitope at different stages after infection with influenza A virus using NP366–374/Db tetramers in combination with a panel of mouse Vß-specific mAbs. This set of mAbs detects 17 out of 24 known Vß subfamilies in mice. As expected, staining of naive Vß8.3+CD8+ splenic T cells with these mAbs gave rise to a highly diverse distribution pattern with relative dominance of Vß5.1/2 and Vß8.1/2 usage (Fig. 2B). After primary infection with PR8 virus, an obvious skewing to Vß8.3 gene segment was observed. As shown in a representative profile by flow cytometric analysis (Fig. 2A), 50–60% of the NP366–374-specific CD8+ splenic T cells used Vß8.3 gene segment during the peak of a primary and a secondary response. Vß8.3 usage dropped only slightly during resting primary and secondary memory stages and is still prominent among the memory pools. In addition, several other Vß gene fragments, including Vß2, Vß4 and Vß10b, were consistently selected in response to the NP366–374 epitope throughout all four immune stages after infection, although their frequencies were much less pronounced compared to that of Vß8.3-expressing cells (Fig. 2B). Note that the percentages of NP366–374-specific CD8+ T cells that expressed Vß2, Vß4 and Vß10b gene segments significantly increased after repeated exposure to the virus. Whereas only 1.7%, 2.5% and 0.5% of the primary cells expressed Vß2, Vß4 and Vß10b gene segments, respectively, the corresponding numbers increased to 14.3%, 9.0% and 12.3% in the resting secondary memory pool for NP366–374-specific CD8+ T cells. Given that the percentage of Vß8.3 usage was relatively stable at each of the four different immune status periods examined here, the data strongly suggest that the diversity of the non-Vß8.3 repertoire for NP366–374 epitope was narrowed after repeated exposure to the virus. The usage of the remaining 13 Vß segments by NP366–374-specific CD8+ T cells was variable and marginal (Fig. 2B).



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Fig. 2. TCR Vß gene segment usage by NP366–374-specific CD8+ T cells. Spleens were harvested after primary or secondary infections or during their respective resting memory stages as indicated. Single cell suspensions were stained with NP366–374/Db tetramer in combination with a panel of mouse Vß-specific mAbs and anti-mouse CD8{alpha} mAb. cells were gated for further analysis of Vß usage. (A) Representative profiles of flow cytometric analysis. The numbers in the upper quadrant of the each dot plot represent the percentage of a given Vß+ cell population among the total cells. (B) Summary of Vß gene segment usage by naive CD8+ T cells versus NP366–374/Db-specific CD8+ T cells obtained at different stages after infection with influenza virus. The numbers in the upper right corner of each individual figure represent the percentages of the total Vß+ cells detected among the total CD8+ T cells. (C) Sequence differences among Vß8.1–8.3 CDR1, CDR2 and 4th loop segments. Amino acid differences in the 4th loop segments are underlined.

 
Together, the data demonstrate that CD8+ T cells expressing Vß8.3 comprise the dominant response within the NP366–374-specific repertoire at all different stages after infection in vivo. Therefore, further analyses were focused on this subpopulation within the NP366–374-specific CD8+ T cell repertoire in the present study. Nevertheless, it will be intriguing to dissect the Vß2, Vß4 and Vß10b repertoire associated with recognition of the NP366–374 epitope in the future.

CDR3ß length distribution of the NP366–374-specific TCR repertoire in individual mice
Analysis of TCR Vß usage provides information on the overall diversity of a T cell repertoire. However, a T cell population expressing a single Vß segment may be heterogeneous with multiple TCRs incorporating different CDR3ß regions. To obtain more detailed information on the NP366–374-specific repertoire, we analyzed the CDR3ß size distribution of T cells ex vivo by spectratyping. The latter is an RT–PCR-based approach that has been successfully used to analyze TCR repertoires from a wide variety of human and mouse materials (25). Antigen-driven clonal expansion of T cell populations skews the distribution of their CDR3 lengths, which is otherwise a normal distribution of variable lengths in naive T cells.

When unfractionated materials were used for the initial analysis, we found significant background peaks in the spectratypes due to co-amplification of irrelevant TCRs. This effectively masked a skewed CDR3 distribution profile in some cases, significantly interfering with the interpretations of the spectratyping results (data not shown). Therefore, we applied a NP366–374/Db tetramer-based cell sorting step to obtain highly pure NP366–374-specific CD8+ T cells as the starting material. This modification has significantly increased the resolution of our approach (data not shown).

As evident from Fig. 3 (upper two panels) and as expected, Vß8.3+CD8+CD44low T cells from two naive B6 mice showed a typical Gaussian-like distribution pattern, indicating a polyclonal (i.e. diverse) repertoire. After primary infection with PR8 virus, a strong selection for both Vß8.3-Jß2.2 and Vß8.3-Jß1.6 combinations, with a nine amino acid length CDR3ß segment, was observed (two middle panels of Fig. 3). Note that these two VDJ combinations were shared by the two individual mice examined. Nevertheless, distinct repertoire distortions were evident between the two individuals after primary infection. For example, compare spectratypes involving Jß1.2, Jß1.5 and Jß2.3 in combination with the Vß8.3 gene segment. Most importantly, we observed a further focusing of Jß gene usage during the secondary response (two lower panels of Fig. 3). In fact, only two Jß gene segments, e.g. Jß2.2 and Jß1.6, were detected. Spectratyping with Vß8.3 and Cß primers independently confirms the evident focusing of the immune response apparent from employing Vß8.3 in conjunction with Jß primers. The remaining Jß spectratyping profiles from the virus-infected animals were not shown, as they were indistinguishable to the corresponding profiles of the two naive mice.



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Fig. 3. CDR3ß length distribution of NP366–374/Db-specific CD8+ T cells. T cells were sorted directly ex vivo from the spleen of two individual B6 mice either on day 10 after a primary infection or on day 7 after a secondary infection with influenza virus. Vß8.3+CD8+CD44low T cells were sorted from two individual naive B6 mice as control. cDNAs were prepared from total RNA extracted from the sorted cells, and then subjected to spectratyping analysis as described in Methods.

 
Together, the data show that primary T cells are highly heterogeneous with regard to their CDR3ß length distribution. However, common or public components, e.g. Vß8.3-Jß2.2 and Vß8.3-Jß1.6 combinations, with a nine amino acid CDR3ß segment, exist among individual repertoires. These two shared components are dramatically enriched within the NP366–374-specific repertoire after a second exposure to the virus.

CDR3ß sequences associated with recognition of the NP366–374/Db complex in individual mice
Spectratyping is a powerful tool for analyzing CDR3 size distributions with a given T cell population. However, CDR3 fragments that are identical in length yet differing in amino acid sequence appear as a single peak in spectratyping profiles. In order to reveal the complexity of the NP366–374 CDR3ß repertoire, we cloned and sequenced CDR3ß regions of TCRs from two individual mice after either primary or secondary infection. A total of 36 clones were sequenced from the sorted -specific T cells after primary infection. Distinct nucleotide sequences and deduced CDR3 amino acid sequences are shown in Table 1. Consistent with the results obtained by spectratyping analyses (Fig. 3), a majority of the 36 clones used Jß2.2 (22/36), and to a lesser extent, Jß1.6 (12/36). In addition, Jß2.5 and Jß2.7 usage were also detected, although in low frequency. When nucleotide sequences were compared, the CDR3ß repertoires of the two animals were highly diverse. Sixteen distinct nucleotide sequences out of 36 sequences examined were recorded. Most were distinct or ‘private’ for the two individuals (nucleotide sequence PN1.1-PN1.6 for mouse #1 and PN2.1-PN2.7 for mouse #2, respectively). However, at least two nucleotide sequences, e.g. sequence CN1 and CN2, were shared by both animals (CN1: 4/20 for mouse #1 versus 5/16 for mouse #2; CN2: 1/20 for mouse #1 versus 1/16 for mouse #2, respectively). When the deduced amino acid sequences of CDR3ß regions were compared, the two individual mice showed a remarkable overlap among their primary CDR3ß repertoire. Three CDR3ß regions, corresponding to amino acid sequence CA1, CA2 and CA3, respectively, were identical in both mice, although their frequencies varied considerably. In addition, distinct amino acid sequences of CDR3ß regions within the primary repertoire were evident between the two animals (sequence PA1.1, and PA1.2 for mouse #1; sequence PA2.1, and PA2.2, PA2.3 for mouse #2, respectively).


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Table 1. Nucleotide and deduced amino acid sequences of TCR CDR3ß junctional regions from ex vivo recovered NP366–374-specific CD8+Vß8.3+ T cells

 
When the NP366–374-specific CDR3ß repertoires were analyzed at the peak of a secondary response to the influenza A virus, the diversity of the repertoire decreased substantially (Table 1). Of a total of 56 independent clones obtained from two individual mice, seven distinct nucleotide sequences of CDR3ß regions were detected, encoding only four different CDR3ß amino acid sequences. Most importantly, two CDR3ß, e.g. amino acid sequence CA2 and CA4, were shared in common among the secondary repertoire of the two individuals with extremely high frequency (sequence CA2: 14/30 for mouse #1 and 21/26 for mouse #2; sequence CA4: 6/30 for mouse #1 and 5/26 for mouse #2). It is important to note that one amino acid sequence of CDR3ß region (sequence CA2) was shared by both the two primary and the two secondary animals. In addition, two additional amino acid sequences of CDR3ß region, e.g. sequence CA1 and CA4, were found in three out of the four animals tested. Consistent with the result from spectratyping analysis (Fig. 3), only Jß2.2 and Jß1.6 usage were detected within the secondary repertoire.

To rule out the possibility that the results described above arose from technical errors generated during the process of DNA cloning and sequencing, cDNA from Vß8.3+CD8+CD44low cells of a naive B6 mouse was amplified by Vß8.3-Cß primers. The purified PCR product was then cloned and sequenced following the same procedure used for the materials obtained from the virus-infected animals. As shown in Table 2, a broad range of Vß8.3-Jß combinations was detected from the 14 sequenced clones. Further, the CDR3ß size distribution was variable, ranging from 6 to 10 amino acids in length. Each clone represented a unique CDR3ß region, both with regard to nucleotide and to amino acid sequence. This result demonstrates that the highly focused NP366–374-specific immune repertoire detected by the same procedure is not methodological but rather reflects the actual frequency of CDR3ß in the immune versus naive repertoire.


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Table 2. TCR CDR3ß junctional regions from CD8+Vß8.3+CD44low naive T cells

 
Together, the data demonstrate a clonal maturation process during transition from primary to memory T cell response against the NP366–374 epitope, whose trend can be clearly seen when CDR3ß sequence data from Table 1 were combined from two individual mice. As shown in Fig. 4, such a maturation process is characterized by a significant decrease of distinct CDR3ß sequences relative to the naive repertoire and a concurrent increase of common CDR3ß regions within the secondary repertoire after a repeat exposure to the influenza virus. Note, for example, that 70% of CDR3ß segments found in the secondary responses contain a common GSN sequence compared to <50% in the primary response. In addition, in the primary response, the GSN, GGN and GAN motifs are more comparably represented than in the secondary repertoire.



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Fig. 4. Clonal maturation within the NP366–374/Db-specific repertoire during transition from primary to memory response. The data from infected mice are combined from the results of two individual animals shown in Table 1. The naive mouse data derive from a single animal. CDR3ß motifs refer to the GXN sequence in combination with Jß2.2 usage found in at least three of four mice examined, where G represents glycine at position 97 in the CDR3ß segment, X is serine, glycine or alanine at position 98, and N is asparagine at position 99.

 
Dissociation of NP366–374/Db tetramer binding from bulk populations of NP366–374-specific CD8+ T cells
The selection for certain TCRs within the NP366–374 repertoire during transition from the primary to the memory response may be a consequence of different TCR affinity or avidity for the pMHC complex. CD8+ T cells expressing TCRs with higher affinity to the NP366–374/Db ligand may be selectively expanded over those with lower affinity TCRs after secondary exposure to the virus. To test this hypothesis, we measured the dissociation rates of the NP366–374/Db tetramer from the bulk populations of NP366–374-specific CD8+ T cells obtained after primary and secondary exposure to influenza A virus, respectively. This assay has been successfully used to measure the avidity of a T cell population with its peptide/MHC ligand (21,26) based on the original observation that the level of epitope/MHC tetramer binding to peptide-specific T hybridomas correlated with the affinity of the TCRs for the relevant peptide/MHC ligands (27).

As shown in Fig. 5(A), dissociation kinetics of the NP366–374/Db tetramer from primary bulk T cell populations varied considerably from mouse to mouse. In contrast, variations were much less pronounced between individual secondary repertoires. This indicates that the TCRs selected in the secondary repertoire are more restricted than the primary repertoire, consistent with the CDR3ß sequencing data (Table 1). The slope of each curve and the half-life of the interactions were calculated for the time intervals indicated, to compare the tetramer dissociation rate between the two groups. As shown in Fig. 5(B), the NP366–374/Db tetramer dissociated relatively faster from the TCRs of the primary bulk population than from those of the secondary repertoire within the first 1 h (the t1/2 was 32.2 min and 47.8 min, respectively, P < 0.0001). However, when the later 1–3 h time interval was compared, the primary and secondary dissociation curves were essentially comparable (t1/2 was 126.0 min versus 130.8 min, respectively, P > 0.7685). Together, the data strongly suggest that a portion of the primary repertoire contains TCRs with relatively high dissociation rate for the NP366–374/Db ligand. Upon secondary viral exposure, this subset of low avidity T cells is absent and high-avidity oligoclonal T cell populations are selectively expanded.



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Fig. 5. Dissociation of NP366–374/Db tetramer from NP366–374-specific, bulk CD8+ T cell populations obtained from the lungs of mice following primary or secondary infection with influenza A virus. The original normalized fluorescence intensities from twelve individual animals in each group were plotted against time elapsed (A). The slopes and the half-lives (t1/2) of the tetramer dissociation at the time intervals indicated were calculated as described elsewhere (21) but employing repeated measures analysis.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
Several earlier studies have contributed to our understanding of TCR repertoire selection to model antigens as well as to infectious agents. Whereas the diversity of the MHC class II-restricted CD4+ T cell repertoire to PCC has been found to be significantly narrowed after a secondary exposure to the antigen (11), a similar phenomenon has not been observed in MHC class I-restricted CD8+ T cell repertoires to a tumor-derived antigen HLA-CW3 (28) or viral pathogens (13,14,16). In the present study, we analyzed the selection process of the TCR Vß repertoire ex vivo during primary and secondary responses to an immunodominant CTL epitope of influenza A virus. Our results demonstrate that the diversity of the secondary repertoire is considerably decreased compared to the primary repertoire in this system, both with regard to the CDR3ß length distribution and CDR3ß sequence. Perhaps more importantly, we found that the reduction in diversity largely resulted from a strong selection of the public components within the repertoire of B6 (H-2b) mice after a secondary viral exposure.

We used PCR-based approaches, e.g. spectratyping, cloning and sequencing of CDR3ß regions, to analyze the molecular features of TCRs associated with recognition of the NP366–374 epitope. Although PCR-based artifacts can be generated from this type of analysis, several arguments speak for the reliability of our results. First, starting materials were obtained from individual mice and the cells were sorted in independent experiments. Second, cDNA materials from these animals were never amplified in consecutive PCR experiments. Third, two common CDR3ß amino acid sequences identified in this study, e.g. sequence CA2 and CA3 (Table 1), have been reported to be associated with NP366–374/Db specificity in a previous independent study using T cell hybridomas (9). Fourth, and most compellingly, CDR3ß regions from the T cells of a naive control mouse did not show highly selective sequence features as observed for the NP366–374 repertoire of the virus-infected animals (Table 2).

Recent studies, which have examined TCR repertoire selection in MHC class I-restricted systems, found no evidence for a more focused repertoire after secondary exposure to the same antigens. At present, we do not know the basis for the different conclusions reached in those studies. Clearly, other antigenic peptides and MHC restriction elements are used in these systems, which may lead to distinct outcomes, but the disparate results may also be reflective of the different methodologies employed. In this regard, it has become clear that analysis of TCR Vß usage alone does not provide sufficient resolution to reveal the clonal complexity of the TCR repertoire. All but one study (12) failed to observe a narrowing of repertoire at the Vß usage level during a recall response. Moreover, although spectratyping is a powerful technique to characterize the CDR3 size distribution patterns of a T cell repertoire, a T cell population with the same CDR3 length could be either extremely diverse or highly homogeneous at its primary amino acid sequence level. Our results show that comprehensive CDR3 sequencing is crucial to reveal the clonal complexity of a TCR repertoire. In this regard, nucleotide sequence analysis was not performed in many of the prior studies addressing this issue in a viral system (1315).

In contrast to our results, Turner et al. did not find narrowing of the secondary repertoire to PA224–233, another Db-restricted immunodominant CTL epitope of influenza A virus in B6 mice, even though comprehensive CDR3ß junctional sequencing was performed on an epitope-specific single cell basis in this recent study (16). Given that both antigenic peptides use the same MHC class I element for presentation, this fundamental difference in immune repertoire selection is highly significant. Results from that same laboratory (24) as well as ourselves herein (Fig. 1) have demonstrated a substantial shift of immunodominance for NP366–374 and PA224–233 epitopes between primary and secondary response to the virus. While both epitopes are co-dominant in the primary response, the NP366–374 clone assumes dominance in the secondary response. Although the reason for this shift is not certain at present, the lack of a typical secondary response to the PA224–233 epitope is not due to: (i) difference in the viral strain growth rate in vivo; (ii) the difference in frequency in the primary memory pool for NP366–374 and PA224–233 (data not shown); (iii) intrinsic inability of PA224–233-specific primary memory cells to proliferate (24); or (iv) significant differences in binding affinity of the two antigenic peptides to Db molecules (18). Instead, a recent elegant study has suggested that differential presentation of the two epitopes by APCs during secondary infection may have contributed to the changing pattern of immunodominance in vivo (29). Thus, it is likely that the secondary response to PA224–233 (Fig. 1B) is largely ‘frozen’ at the level of the rather diverse primary PA224–233 memory pool due to insufficient subsequent clonal expansion caused by limited epitope presentation after a secondary viral challenge. The highly diverse TCR repertoire to PA224–233 identified by Turner et al. (16) is consistent with this notion. Together, the results presented here (Fig. 1) strongly suggest that an antigen-driven clonal expansion is critical in order for TCR repertoire selection to take place.

Although only the TCR Vß repertoire was analyzed in the present study, we are fully aware of the potential critical contributions of the TCR V{alpha} domain in recognition of the NP366–374/Db ligand and selection of a complete {alpha}ß TCR repertoire in response to this CTL epitope, as clearly suggested by several recent studies in both mouse and human systems (5,30,31). We indeed have obtained some interesting preliminary data indicating that the dominant Vß8.3 gene segment appears to be paired with more diverse V{alpha} gene segments within a secondary NP366–374-specific repertoire (data not shown). Detailed analysis is currently underway in our lab to reveal the molecular features of the TCR V{alpha} repertoire specific for the NP366–374/Db ligand.

Using spectratyping techniques to characterize the murine T cell response to hen egg lysozyme (HEL), Cibotti et al. first observed that T cell responses to an immunodominant epitope of a protein antigen involve a public Vß domain shared by all animals tested, and a private Vß domain unique to individual animals (20). This observation was subsequently confirmed by Lawson et al. who found that influenza antigen exposure selected a dominant Vß17 TCR in the human CD8+ CTL T cell response (32). However, this phenomenon was not observed when the molecular features of CDR3ß regions for the xenogenic model antigen, HLA-CW3, were thoroughly examined (33). These workers found an extremely diverse TCR repertoire in response to immunization with HLA-CW3 such that each mouse developed distinct or private TCR signatures.

In the present study, we observed public components to the Db-restricted influenza virus NP366–374 epitope involving all three elements of TCR ß variable domains. First, the Vß8.3 gene segment usage is the signature response that can be found in all animals tested (Fig. 2). Second, preferential Jß2.2, and to a lesser extent, Jß1.6 usage, was reproducibly detected by the spectratyping assay in each individual (Fig. 3). Third, common amino acid sequences of the CDR3ß region (comparing D segments and somatically generated junctional segments) were found in the primary repertoire and are remarkably enriched among individual animals after secondary viral challenge (Table 1). On the other hand, private, animal unique components were equally apparent in the present study (Fig. 3 and Table 1). Together, our data clearly demonstrate that both public and private responses exist within the immune repertoire of individual B6 animals in response to the NP366–374 epitope of the influenza virus. At present, we do not know about the generality of our findings for most antigen-specific TCR repertoires, as only a limited number of studies have been conducted. Nonetheless, it appears likely that for those epitopes dominating both primary and secondary responses, similar repertoire phenomena will be observed.

Selection for a limited number of germline-encoded TCR V gene segments has been noted following the contact of host CD8+ T cells with a variety of non-infectious and infectious antigens. In the present study, we also found that CD8+ T cells specific for the NP366–374 epitope of the influenza A virus preferentially expressed the TCR Vß8.3 gene segment. It is not clear which factors determine the highly biased nature of Vß gene usage during onset of a T cell response triggered by a particular antigenic peptide. The fact that Vß8.3, but not Vß8.1 or Vß8.2 alleles, is predominantly used in response to the NP366–374 epitope (Fig. 2), suggests that the relatively minor variability within the germline V segment encoded recognition surface including CDR1, CDR2 regions and/or the fourth loop of the TCR molecule might have made a major contribution to this selection process. As shown in Fig. 2(C), the amino acid sequence of CDR1 and CDR2 loops of the Vß8.3 segment are similar to those of Vß8.1 and Vß8.2 alleles, excluding residues therein. However, the fourth loop of Vß8.3 is different from that of Vß8.1 or Vß8.2 at three amino acid positions, e.g. threonines at positions 82 and 83 and aspartic acid at position 86 for Vß8.3, whereas proline, serine and asparagine are at the corresponding positions for both Vß8.1 and Vß8.2 alleles. Such sequence differences, most notably the presence or absence of proline, on the fourth loop of the Vß8 segment might have significant impact on the conformation of the nearby CDR1 and CDR2 loops, thus indirectly influencing the docking of the TCR to the NP366–374/Db ligand. In this regard, conserved CD4+ T cell-derived TCR Vß gene usage in response to a human HLA-DR-restricted HA epitope of influenza virus appears to involve charged interactions between three lysines of the antigenic peptide and acidic residues within the CDR1ß region of the TCR (34). Therefore, it is likely that conserved amino acid residues exist in the germline-encoded regions of each TCR Vß subfamily member that favor key contacts with residue(s) of a given set of peptide-bound MHC complexes, resulting in biased Vß segment usage. Incorporation of such immunoprotective features of immune recognition into the germline then allows for ready selection of useful TCRs that can be further improved upon by the CDR3 selection phenomena described herein.

Consistent with this possibility, tetramer dissociation data (Fig. 5) show that the initial decay of NP366–374/Db tetramer staining on the surface of the primary Vß8.3+ T cell populations was significantly faster relative to the secondary Vß8.3+ T cell populations. Due to low frequency of the NP366–374-specific CD8+ T cells in a resting memory pool after a primary infection with the influenza virus (typically ~0.3% among total CD8+ T cells in spleen), it is unfeasible to examine the diversity and affinity of a primary NP366–374 memory pool at this stage. Thus, the current data do not formally exclude the possibility that primary memory T cells with high affinity have been already selected after initial infection. Nevertheless, this finding clearly indicates that a proportion of primary cells expressing higher dissociation rates for the NP366–374/Db complex are selected against during the secondary response. Thus, although selection of the TCR repertoire occurs after the first antigen exposure, continued narrowing of the repertoire takes place upon successive antigen contact. Such a qualitative change may contribute to the efficacy of T cell memory, allowing a response to secondary antigenic challenge with a significantly enhanced magnitude and accelerated kinetics, thereby improving protective immunity. In this regard, it will be very interesting to compare the diversity and complexity of the NP366–374-specific TCR repertoire in lung and in spleen in the future.

Together, our data support an affinity maturation model of TCR repertoire selection (35). Unlike the stochastic model, which proposes that selection of TCR repertoire during immune response to a peptide antigen is random, the experiments presented here have documented a clear selection process for certain TCRs during the transition from primary effectors to memory effectors in murine CD8+ T cell responses to an immunodominant CTL epitope of influenza virus. To our knowledge, this represents the first example of continued narrowing of the TCR repertoire after secondary exposure to a viral pathogen. This observation is consistent with the results obtained in a CD4+ T cell, MHC class II-restricted system involving the soluble pigeon cytochrome c protein (11). A similar conclusion was inferred in a bacterial system by Busch et al. (12), who observed a trend toward selective expansion of T cells bearing specific Vß gene segments after a secondary bacterial challenge. Together, these studies clearly document an antigen-driven selection process of TCR repertoire upon successive antigen exposures. The extent to which this ‘narrowing’ feature will be shared by TCRs directed at other specificities remains to be demonstrated but given competition for antigen among immune cells, it is likely that survival of the fittest TCR-expressing cells will win out resulting in immune focusing in general.


    Note added in proof
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 
After this work was completed, an independent study from Kedzierska et al. has been published (36). These workers also observed a conserved TCR usage in primary and secondary responses of C57BL/6 mice to the same Db restricted, immunodominant CTL epitope of the influenza virus, e.g. NP366–374. Most importantly, the same ‘public’ Vß-Jß usage and CDR 3ß sequences were identified independently in both studies.


    Acknowledgements
 
This work was supported by National Institutes of Health Grant AI50900 and an award from the Molecular Immunology Foundation. We thank Dr Rob Meijers for molecular modeling insights, Dr Haesook Kim for statistical advice and Drs Linda Clayton, Jonathan Duke-Cohan and Anne Marie Moody for critical review of the manuscript.


    Abbreviations
 
AICD   activation-induced cell death
CDR1   complementarity-determining region 1
CTL   cytotoxic T lymphocyte
HEL   hen egg lysozyme
MLN   mediastinal lymph node
PB   peripheral blood
PCC   pigeon cytochrome c
TCR   T cell receptor

    Notes
 
Transmitting editor: S. Koyasu

Received 2 June 2004, accepted 5 August 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Note added in proof
 References
 

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