HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bour, H. Articles by MacDonald, H. R. Search for Related Content PubMed PubMed Citation Articles by Bour, H. Articles by MacDonald, H. R.

Dramatic Influence of Vß Gene Polymorphism on an Antigen-Specific CD8⁺ T Cell Response In Vivo

Hélène Bour^*, Olivier Michielin, Philippe Bousso, Jean-Charles Cerottini^* and H. Robson MacDonald^1,*

^* Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; Le Bel Institute, Louis Pasteur University, Strasbourg, France; and Laboratory of Molecular Biology of the Gene, Pasteur Institute, Institut National de la Santé et de la Recherche Médicale U277, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
According to recent crystallographic studies, the TCR-ß contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1ß (CDR1ß) and the hypervariable (D)J-encoded CDR3ß and CDR3 domains. To evaluate directly the relative importance of CDR1ß polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic Vß^a and Vß^b mouse strains that differ by a CDR1 polymorphism in the Vß10 gene segment. The Vß10-restricted CD8⁺ T cell response to a defined immunodominant epitope was dramatically reduced in Vß^a compared with Vß^b mice, as measured either by the expansion of Vß10⁺ cells or by the binding of MHC-peptide tetramers. These data indicate that Vß polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1ß-peptide interactions.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recognition of a foreign Ag by T cells is achieved through interaction of the TCR with an antigenic peptide presented by MHC molecules on the surface of APCs. Three hypervariable complementarity-determining regions (CDRs),² named CDR1, CDR2, and CDR3 have been defined on the TCR - and ß-chains (1, 2). CDR3, which encompasses the V(D)J junctional area, is the most variable region and was thus predicted to contact the antigenic peptide, whereas the V gene segment-encoded CDR1 and CDR2 were predicted to interact mainly with the MHC molecule. However, recent crystallographic studies of several TCR/peptide/MHC trimolecular complexes have identified direct contact of both CDR1 and CDR3 of the - and ß-chains with the antigenic peptide (3, 4, 5). Because CDR1 sequences are polymorphic in mice and humans (6, 7), it follows that this polymorphism may be important for the fine specificity of T cell responses to antigenic peptides.

To investigate this possibility, we have used a well-characterized in vivo model of a Vß-restricted Ag-specific CD8⁺ T cell response. DBA/2 mice (Vß^b haplotype) injected i.p. with syngeneic P815 tumor cells (H-2^d) transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8⁺ T cells exclusively expressing the Vß10 segment (8). The specific cytotoxic activity against the immunodominant peptide CW3_170–179 presented by the H-2K^d molecule was found exclusively in the CD8⁺ CD62 ligand (CD62L)^- Vß10⁺ population (8, 9), and the CW3-specific CD8⁺ Vß10⁺ T cell expansion could be readily monitored by flow microfluorometry in peripheral lymphoid organs, in the liver, and in PBLs (10). Furthermore, the characterization of CW3-specific TCRs on CTL clones and by single-cell PCR analysis suggested that the CW3-specific repertoire size is limited to 15–20 clones per mouse, and that all of the clones displayed common characteristics, including an exclusive usage of Vß10 and J35 (previously called JpHDS58) segments, a preferential Jß and V segment usage, and a conserved CDR3 length of 6 and 9 aa in both the ß- and -chains, respectively (11, 12).

A coding sequence polymorphism has been described in the Vß10 gene segment between the Vß^a and Vß^b haplotypes (13). Indeed, comparison of the coding sequence of all known Vß genes between the Vß^a and Vß^b haplotypes showed that Vß10 alleles are the most polymorphic among the Vß genes analyzed, with six amino acid differences at the protein level (Fig. 1). Among these differences, three amino acid changes are located in or in the immediate vicinity of CDR1 of the Vß10 segment and markedly differ with respect to charge or polarity, namely Lys-Glu (Vß^b-Vß^a) at position 24, Gly-Asp at position 28, and Asp-Asn at position 30. In contrast, CDR2 remains unchanged between the two alleles. Thus, this model represents a unique opportunity to study the influence of a naturally occurring ß-chain polymorphism on an Ag-specific response in vivo. In this study, we have compared the CW3-specific CD8⁺ T cell response in DBA/2 mice, which are congenic for the Vß^a and Vß^b alleles. Our results show that the CW3-specific CD8⁺ Vß10⁺ T cell response is dramatically reduced in DBA/2 mice of the Vß^a haplotype, suggesting that Vß gene polymorphism has a major impact on Ag recognition and TCR repertoire selection.

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Polymorphic residues distinguishing the Vß10 gene between the Vßa and Vßb haplotypes. Single-letter amino acid symbols are used. The upper line shows the Vß10 sequence in the Vßa haplotype. On the lower line, identical residues in the Vß10b allele are indicated by a dash; differences are highlighted. CDR1 and CDR2 are indicated by underlining. Amino acid sequences and the position of the CDRs are as reported by Arden et al. (6). The dots below the sequences are located every 10 aa.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Adult female DBA/2 mice (Vß^b haplotype) were purchased from Harlan Olac (Bicester, U.K.). DBA/2 mice of the Vß^a haplotype were kindly provided by Dr. A. Livingstone (Basel Institute for Immunology, Basel, Switzerland) and were maintained in our own animal facilities. These mice carry the TCR Vß^a locus derived from C57L mice and were backcrossed to DBA/2 for 15 generations. DBA/2 mice of the Vß^b or Vß^a haplotype were injected i.p. with 10⁷ P815-CW3 cells (14).

At 2 wk postimmunization, mice were bled by the tail vein and PBLs were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala, Sweden). For H-2K^d-CW3 tetramer staining, PBLs were further depleted of remaining RBCs by a 1-min treatment with NH₄Cl. Intrahepatic lymphocytes were isolated using Percoll gradient centrifugation as described previously (15).

Intrahepatic lymphocytes were tested directly for the lysis of P815 cells, P815 cells coated with the CW3_170–179 peptide, and P815-CW3 transfectant cells, as described previously (16). Indeed, freshly isolated intrahepatic lymphocytes from HLA-CW3 immune mice have been shown to be very potent with regard to their ability to exert CW3-specific lysis without sorting or further manipulation (10 and our unpublished observations).

Flow cytometry analysis

Triple staining was performed on Vß^b PBLs with FITC-conjugated anti-CD62L (Mel-14, prepared in our laboratory), phycoerythrin (PE)-conjugated anti-CD8 (53.6.7; Boehringer Mannheim, Mannheim, Germany), and biotinylated anti-Vß10^b (B21.5, prepared in our laboratory) revealed with streptavidin-tricolor (Caltag Laboratories, San Francisco, CA). PBLs recovered from Vß^a mice were labeled with unconjugated anti-Vß10^a (KT10a, generously provided by Dr. Tomonari, Fukui, Japan) (17) revealed with FITC-conjugated goat anti-rat Ig (Caltag). After blockade with rat IgG, PBLs were subsequently stained with PE-conjugated anti-CD8 and biotinylated anti-CD62L (Mel-14, prepared in our laboratory) revealed with streptavidin-tricolor. In some experiments, PBLs were also stained with FITC-conjugated anti-Vß2 (B2O.6.5), FITC-conjugated anti-Vß14 (14.2), or biotinylated anti-Vß4 (KT4.10) (all prepared in our laboratory). Samples were analyzed on a FACScan equipped with Lysis II software (Becton Dickinson, San Jose, CA).

Biotinylated complexes of H-2K^d-CW3 peptide 170–179 (RYLKNGKETL) were produced as described by Bousso et al. (18). To generate the H-2K^d-CW3 tetrameric complexes, the biotinylated monomers were mixed with streptavidin-tricolor at a molar ratio of 4:1. Four-color stainings were performed for analysis with H-2K^d-CW3 tetrameric complexes. A total of 2 x 10⁵ cells were incubated at 4°C for 1 h with tricolor-labeled H-2K^d-CW3 tetramers, washed twice, and stained as described above with the following mAbs: anti-Vß10^a revealed with goat anti-rat Ig FITC, FITC-conjugated anti-Vß10^b or anti-Vß2-FITC associated with anti-CD62L-PE (Caltag), and anti-CD8-APC (53.6.7. PharMingen, San Diego, CA). Samples were analyzed on a FACScalibur equipped with CellQuest software (Becton Dickinson).

Generation of CTL clones

Vß^a mice were killed 2 wk after an i.p. injection of P815-CW3 cells. Single-cell suspensions of splenocytes were prepared by standard procedures and purified by nylon wool columns. A total of 20 x 10⁶ purified immune splenocytes were cultured with 1 x 10⁶ irradiated P815-CW3 tumor cells and 10 x 10⁶ irradiated naive T cell-depleted splenocytes as feeder cells in 10 ml DMEM (Life Technologies, Paisley, U.K.) supplemented with 2 mM L-glutamine, 1 mM HEPES, 3 x 10^-5 M 2-ME, and 5% heat-inactivated FCS (Irvine Scientific, Santa Ana, CA). Mixed lymphocyte tumor cultures were performed for 10 days, the last 3 days in the presence of 30 U/ml IL-2 (EL-4 cell-conditioned supernatant, prepared in our laboratory), and CTL clones were derived by limiting dilution as described previously (14, 19). CTL clones were screened for their ability to lyse P815-CW3 but not P815 targets in a 4-h ⁵¹Cr release assay (16).

PCR amplification and sequencing of TCR - and ß-chains

Total RNA was extracted from Vß^a CTL clones using an RNeasy mini kit (Qiagen AG, Basel, Switzerland). A single-stranded cDNA template was prepared by reverse transcription using 2 µg of total RNA, 200 nmol of oligo(dT) primer, 160 nmol of deoxynucleoside triphosphate, and 50 U of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) for 90 min at 42°C in a 40-µl final volume. For cDNA amplification by PCR, we used the Vß, Cß, V, and C primers reported previously (20), with the exception of the newly designed primer specific for Vß10^a (5'-ATCAAGTCTGTAGAGCTGGAGGAC-3'). Aliquots of the cDNA were amplified in a 50-µl final volume using Vß-Cß or V-C primer combinations (all at a final concentration of 400 nM) with a reaction mixture containing 800 µM of deoxynucleoside triphosphate, 1.5 mM MgCl₂, PCR buffer, and 1.8 U Expand high fidelity polymerase (Boehringer Mannheim). A total of 40 cycles, each at 94°C for 30 s, 56°C for 45 s, and 72°C for 45 s, were performed in a Biometra thermocycler (Biometra, Tampa, FL). PCR products were sequenced using a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Amersham, Buckinghamshire, U.K.) according to the manufacturer’s instructions and analyzed in a LI-COR DNA sequencer (MWG Biotec, Munchenstein, Switzerland).

Molecular modeling

A homology model of the 1C8 TCR/CW3/H-2K^d complex was built based on all of the available TCR-ligand crystal structures (3, 4, 5, 21) and on a structure of the H-2K^b vesicular stomatitis virus peptide (22). This last structure was chosen because its amino and carboxy termini are identical with the CW3_170–179 peptide. The choice of the Vß^b 1C8 TCR (11) was dictated by its -chain usage, which is identical at the amino acid level with the -chain expressed by the Vß^a clone 1.1.C7 (Fig. 6D) recognizing the CW3_170–179 peptide. A chain-by-chain multiple alignment was performed between the sequences using a dynamic programming method (23) implemented in the MODELLER program (24). The alignment was optimized in the peptide region to increase the sequence identity with the H-2K^b vesicular stomatitus virus peptide (22) structure. Based on the multiple alignment, a heavy atom model of the complex was then built using the MODELLER program. Global optimization of the model was achieved through sequential simulated annealing and conjugate gradient energy minimization calculations within the MODELLER program. All of the residues of the Vß CDR1 as well as residues 7–9 of the CW3 peptide were subsequently refined using simulated annealing techniques with the rest of the fixed structure. The conformer with the lowest energy that was consistent with the experimental data (25, 26) was kept. Hydrogen atoms were added using the CHARMM 25 program (27) with the all-atom PARAM 22 parameter set (28). The orientation of the 1C8 TCR with respect to H-2K^d in the model complex is similar to that of the template structures (3, 4, 5, 21), as expected from the procedure used. It is noteworthy that our model predicts an interaction between the highly conserved residues TCR CDR1ß His²⁹ and H-2K^d Ala¹⁵⁰, as described by Garcia et al. in the crystal structure of the 2C TCR/dEV8/K^b complex (5), thus further supporting the CDR1 conformation prediction in our model. The C root mean square deviation between the model complex and the 2C TCR/dEV8/K^b crystal (5) was 1.43 Å (for the V and Vß domain of the TCR, the 1 and 2 domain of the MHC, and the peptide), with the major deviation arising in the region of the CDRs (the C root mean square deviation per residue was <1 Å over the ß-sheet framework and on the order of 4–5 Å in the CDR).

View larger version (36K): [in this window] [in a new window]   FIGURE 6. TCR ß and junctional nucleotide and amino acid sequences of Vßa CTL clones. A and B, Nucleotide sequences of ß- and -chain junctional regions. The Vß, V, Jß, and J segment usages are reported. The nomenclature and sequences for the Vß and V segments are as described by Arden et al. (6). Jß sequences are from Chien et al. (43) and Gascoigne et al. (44), and J sequences are from Koop et al. (45). Usage of Vß2, Vß4, and Vß10a was confirmed by surface staining with corresponding mAbs. Sequence homology with Vßb clones is reported, and nucleotide differences are indicated in bold. C and D, Deduced amino acid sequences of ß- and -chain junctional regions are reported in single-letter amino acid code. Presumed CDR3 regions are putatively supported by two framework branches (FW). The CTL clones for which the transcript can be unambiguously assessed to encode the functional -chain (due to the presence of a second nonproductive rearrangement) are indicated in italic. Identities with Vßb CTL clones at the amino acid level are indicated. Recognition of the CW3170–179 peptide is indicated for each clone.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Confirming previous reports from our laboratory (8, 9, 10), the results presented in Fig. 2 and Table I show that an i.p. injection of P815-CW3 tumor cells in DBA/2 mice (Vß^b haplotype) is followed by a very strong expansion of CW3-specific CD8⁺ Vß10⁺ T cells leading to tumor rejection in 12 of 12 mice. Indeed, 74% of activated (CD62L^-) CD8⁺ PBLs expressed Vß10 in immune DBA/2 mice compared with 10% in the CD62L⁺ CD8⁺ subset of immune (Table I) or naive (9, 10) animals. By contrast, P815-CW3 tumor cells were not rejected in 5 of 29 DBA/2 mice of the Vß^a haplotype, and the mice died rapidly of tumor growth (Table I). Furthermore, as shown in Fig. 2, only 4 of the 24 remaining DBA/2 Vß^a mice showed an expansion of Vß10^a+ cells in the CD62L^- CD8⁺ T cell population. In three of these four mice, the percentage of Vß10^a+ cells reached 30–40% of the CD8⁺ CD62L^- population compared with a control level of 20% on average in the CD8⁺ CD62L⁺ population in immune (20.3 ± 0.9, Table I) and naive (19.8 ± 0.7, data not shown) DBA/2 Vß^a mice. Only one Vß^a mouse displayed a level of Vß10⁺ cells similar to what is normally observed in Vß^b mice, with 65% of Vß10^a+ cells in the CD8⁺ CD62L^- population. Statistical analysis of the results obtained in the 24 Vß^a mice showed no significant difference (Student’s t test, p > 0.5) in the level of Vß10^a+ cells in the CD8⁺ CD62L^- population compared with the control CD8⁺ CD62L⁺ population (Table I). Furthermore, as shown in Table I, the percentage of CD62L^- cells in the CD8⁺ population in Vß^a mice was more variable and was significantly lower than in Vß^b mice (Student’s t test, p < 0.005); consequently, the percentage of CD8⁺ T cells in immune PBLs (10.5 ± 1.7%) did not significantly differ from naive Vß^a mice (8.9 ± 1.5%).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Magnitude of the CD8+ Vß10+ response in Vßa or Vßb mice injected with P815-CW3 cells. DBA/2 mice of the Vßa or Vßb haplotype were injected i.p. with P815-CW3 tumor cells. At 2 wk postimmunization, PBLs were triple-stained with mAbs against CD8, CD62L, and Vß10a or Vß10b, respectively. The percentage of Vß10a+ cells in the CD8+ CD62L- population is indicated for 24 individual Vßa mice. The control level of Vß10a+ cells in the CD8+ CD62L+ population in Vßa mice as well as the results obtained in Vßb mice (n = 12) are expressed as mean percentages ± SD.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Vß repertoire of the anti-HLA-CW3 CD8 response in Vßa mice. PBLs were recovered from DBA/2 mice of the Vßa haplotype 2 wk after an i.p. injection of P815-CW3 cells and were triple-stained separately with mAbs against CD8, CD62L, and either Vß2, Vß4, Vß10a, or Vß14. Vßb mice were immunized and analyzed simultaneously as controls, and results are shown on the left. The percentage of Vß+ cells in the CD8+ CD62L- population is indicated for individual mice. Horizontal lines represent the mean value of each group. Control levels of each Vß in the CD8+ CD62L+ population are indicated below the graph and are expressed as mean percentages ± SD.

The Vß^a locus of DBA/2 Vß^a mice was introduced from C57L mice by backcrossing to the DBA/2 strain for 15 generations. Despite the extensive backcrossing, the immune response against P815-CW3 cells in DBA/2 Vß^a mice could still be due to an influence of the genetic background. In particular, the presence of minor Ags, which could give rise to new peptide-MHC complexes in the thymus of DBA/2 Vß^a mice, cannot be formally excluded. Such peptides could theoretically negatively select CW3-specific T cells, thus accounting for the lack of a Vß10 response in HLA-CW3 immune Vß^a mice. Alternatively, the reduced CD8⁺ Vß10⁺ response could be secondary to an altered positive selection of CW3-specific T cells due to the absence of positively selecting (Vß^b-derived) peptides in the thymus of the DBA/2 Vß^a mice.

To rule out these possibilities, we crossed DBA/2 mice of the Vß^a and Vß^b haplotypes and analyzed the expansion of Vß10^b+ and Vß10^a+ T cells in (Vß^a x Vß^b)F₁ mice after an i.p. injection of P815-CW3 cells. All of the (Vß^a x Vß^b)F₁ mice rejected the P815-CW3 tumor cells. As shown in Fig. 4, CD8⁺ Vß10^b+ T cells expanded in all of the HLA-CW3 immune (Vß^a x Vß^b)F₁ mice, with the percentage of Vß10^b+ T cells in the CD8⁺ CD62L^- population reaching 80% in several F₁ mice. Although slightly weaker than in Vß^b mice, the strong expansion of Vß10^b+ cells in HLA-CW3 immune (Vß^a x Vß^b)F₁ mice showed that CW3-specific CD8⁺ T cells were not negatively selected in the thymus and were present and fully functional in the periphery in a genetic background shared with the DBA/2 Vß^a mice. However, as shown in Fig. 4, no expansion of CD8⁺ Vß10^a+ cells was detected in (Vß^a x Vß^b)F₁ mice after immunization with P815-CW3 cells. This result ruled out both a defective peptide pool available for positive selection and the presence of negatively selecting peptides as an explanation for the reduced CW3-specific CD8⁺ Vß10⁺ T cell response in Vß^a mice. In all F₁ mice, the percentage of Vß10^a+ cells in the CD8⁺ CD62L^- population even decreased compared with the control level in the CD8⁺ CD62L⁺ population, presumably as a consequence of the Vß10^b+ cell expansion. Indeed, the percentages of Vß2⁺, Vß4⁺, and Vß14⁺ cells similarly decreased in the CD8⁺ CD62L^- population of F₁ mice compared with control values (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Relative expansion of Vß10a vs Vß10b CD8+ T cells following immunization of (Vßa x Vßb)F1 mice with P815-CW3 cells. (Vßa x Vßb)F1 mice were injected i.p. with P815-CW3 tumor cells. At 2 wk postimmunization, PBLs were separately stained with mAbs against CD8, CD62L, and either Vß10a or Vß10b (KT10a and B21.5 mAbs are specific, respectively, for Vß10a and Vß10b, and do not cross-react) (17). The percentage of Vß10a+ and Vß10b+ cells in the CD8+ CD62L-population is indicated for individual F1 mice. Control levels of Vß10a+ and Vß10b+ cells in the CD8+ CD62L+ population are shown at the left of the graph and are expressed as mean percentages ± SD (n = 12).

As previously mentioned, several Vß genes are deleted in mice of the Vß^a haplotype. Therefore, the lack of a CD8⁺ Vß10⁺ response in HLA-CW3 immune Vß^a mice could be explained by the absence of CD4 help, due to the deletion of Vß segments in Vß^a mice that are required for an HLA-CW3-specific helper response. However, several lines of evidence argue against this possibility. We have shown previously that CD4 help was necessary for the development of the CW3-specific CD8⁺ Vß10⁺ T cell response and tumor rejection after DBA/2 Vß^b mice were injected i.p. with P815-CW3 tumor cells (9). The rejection of P815-CW3 cells (injected i.p.) in 24 of 29 Vß^a mice consequently suggested that CD4 help is available. More directly, CD8⁺ Vß10^a+ T cells did not expand in (Vß^a x Vß^b)F₁ mice (Fig. 4) despite the fact that one copy of the Vß^b gene locus was present and sufficient to allow the expansion of CD8⁺ Vß10^b+ T cells (Fig. 4). Finally, we have shown that the CW3-specific CD8 response in Vß^b mice can develop in the absence of CD4⁺ T cell help when P815-CW3 tumor cells are injected intradermally (i.d.) (9). Thus, Vß^a mice were injected i.p. or i.d. with P815-CW3 cells, and the Vß10^a+ T cell expansion was compared in the two groups. Results showed that the percentage of Vß10^a+ cells in the CD8⁺ CD62L^- population after an i.d. injection of P815-CW3 cells in Vß^a mice was similar to what was obtained after an i.p. injection (26.2 ± 8.7% vs 21.7 ± 2.0%, respectively; data not shown). The absence of an expansion of CD8⁺ Vß10^a+ cells in a situation in which CD4 help was not required thus argues against a potential deficiency in CD4 help as an explanation for the altered CW3-specific CD8⁺ Vß10⁺ response in Vß^a mice. Collectively, these data rule out the possibility that the absence of a CW3-specific Vß10 response in Vß^a mice is due to a lack of CD4⁺ T cell help.

Specificity of the anti-HLA-CW3 CD8 response in Vß^a mice

We have shown previously in Vß^b mice that CD8⁺ Vß10⁺ T cells specifically recognize the immunodominant peptide CW3_170–179 presented by H-2K^d molecules (8, 14, 19). To our knowledge, no other H-2^d-restricted epitope has been described in the HLA-CW3 molecule. Therefore, we used H-2K^d-CW3 (170–179) tetrameric complexes to further characterize the Vß usage of CW3-specific T cells in Vß^a mice. As shown in Fig. 5A, left panel, in PBLs from HLA-CW3 immune Vß^b mice, staining with H-2K^d-CW3 tetrameric complexes correlated well with the Vß10 staining in the CD8⁺ CD62L^- population. Indeed, the percentage of Vß10^b+ cells in the CD8⁺ CD62L^- tetramer⁺ population was >95% in all of the mice tested, with a mean value of 97.1 ± 0.9%; this finding is in accordance with our report that only CD8⁺ CD62L^- Vß10^b+ T cells from HLA-CW3 immune mice could lyse target cells coated with the CW3 peptide (9). H-2K^d-CW3 tetramer⁺ Vß10^b+ cells accounted for an average of 57.1 ± 10.5% of the CD8⁺ CD62L^- population in nine HLA-CW3 immune Vß^b mice. It is noteworthy that this value is in the same range as the percentage of Ag-specific CD8⁺ T cells detected with MHC-peptide tetramers during viral infections (31, 32, 33, 34). As expected, H-2K^d-CW3 tetramer⁺ cells were restricted to the CD8⁺ CD62L^- population and could not be detected in the control CD8⁺ CD62L⁺ population (data not shown). Interestingly, in all HLA-CW3 immune Vß^b mice, 15–25% of Vß10⁺ cells in the CD8⁺ CD62L^- population (mean value of 20.2 ± 3.8%) were not labeled with H-2K^d-CW3 (170–179) tetrameric complexes. Whether this population of activated CD8⁺ Vß10⁺ H-2K^d-CW3 tetramer^- cells reflects bystander activation or expresses CW3-specific TCRs with an affinity too low to bind H-2K^d-CW3 tetramers, as described previously for CD4⁺ T cell hybridomas (35), remains to be determined. Finally, steric hindrance upon double staining with anti-Vß10^b mAb and H-2K^d-CW3 tetramers was ruled out by a control staining in which an irrelevant anti-Vß mAb (Vß2) was used in conjunction with H-2K^d-CW3 tetramers. As expected, the percentage of CD8⁺ CD62L^- cells stained with H-2K^d-CW3 tetramers remained unaltered (47%), but no double-positive (tetramer⁺ Vß2⁺) cells could be detected using anti-Vß2 mAb (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Specificity of the anti-HLA-CW3 CD8+ response in Vßa mice. A, Staining of HLA-CW3 immune PBLs with H-2Kd-CW3 170–179 tetrameric complexes. HLA-CW3 immune PBLs from Vßb (left panel) or Vßa (middle and right panels) mice were subjected to four-color staining with H-2Kd-CW3 170–179 tetrameric complexes and mAbs against CD8, CD62L, and Vß10b or Vß10a, respectively. Results are presented as cytograms of H-2Kd-CW3 tetramer vs Vß10 staining in the CD8+ CD62L- population, and the percentages of single- and double-positive cells are indicated. The results presented in the left panel are representative of nine Vßb mice; the results obtained for Vßa mouse 3 are representative of 11 Vßa mice. B, Ex vivo lysis of target cells coated with the CW3170–179 peptide. Intrahepatic lymphocytes isolated from HLA-CW3 immune Vßb or Vßa mice were tested directly for the lysis of P815 target cells coated with the indicated concentration of CW3170–179 peptide. The E:T ratio (calculated according to the percentage of CD8+ CD62L- T cells among intrahepatic lymphocytes) was 10:1 in all cases. The percentages of specific lysis of P815 cells coated with the CW3 peptide were normalized to the lysis of P815 cells in the presence of anti-CD3 (2C11) mAb at the same E:T ratio, defined as 100% lysis (the absolute values for the lysis of P815 plus 2C11 were 84%, 76%, 36%, and 46% for the Vßb mouse and for Vßa mice 1, 2, and 3, respectively). C, Ex vivo lysis of P815-CW3 cells. Intrahepatic lymphocytes were assayed directly for cytolytic activity on P815 () or P815-CW3 () target cells. The indicated E:T ratios were calculated according to the percentage of CD8+ CD62L- T cells among intrahepatic lymphocytes.

As mentioned above, it has been shown in CD4⁺ T cell hybridomas that there is a direct correlation between the level of binding of MHC-peptide tetrameric complexes and TCR affinity for its ligand (35). Therefore, to determine whether the absence of H-2K^d-CW3 tetramer⁺ cells in most HLA-CW3 immune Vß^a mice was due to a low affinity of CW3-specific Vß^a TCRs or to a low number of CW3-specific T cells, we compared staining with H-2K^d-CW3 tetramers and recognition of the CW3 peptide in a functional assay. As shown in Fig. 5B, staining with the H-2K^d-CW3 tetrameric complexes correlated directly with ex vivo lysis of target cells coated with the CW3 peptide in HLA-CW3 immune Vß^b and Vß^a mice. Indeed, even in Vß^a mouse 2, in which only 10% of activated CD8⁺ T cells were H-2K^d-CW3 tetramer⁺, target cells coated with the CW3 peptide were lysed with a similar dose-response curve as that seen for Vß^b mice and for Vß^a mouse 1. Conversely, the absence of H-2K^d-CW3 tetramer staining in Vß^a mouse 3 paralleled the absence of lysis of target cells coated with the CW3 peptide. In contrast, P815 cells transfected with the HLA-CW3 gene were efficiently lysed in all Vß^a mice (Fig. 5C) independently of H-2K^d-CW3 tetramer staining. These data strongly suggest that the rejection of P815-CW3 tumor cells in most Vß^a mice is due to the recognition of epitope(s) other than the peptide 170–179 in the HLA-CW3 molecule.

Repertoire of the anti-HLA-CW3 CD8 response in Vß^a mice

CTL clones were derived from HLA-CW3 immune Vß^a mice to compare the sequences of the TCR - and ß-chains with what has been described in Vß^b mice (11, 12). Seven CTL clones specific for the CW3_170–179 peptide and five CTL clones recognizing P815-CW3 transfectants but not the CW3_170–179 peptide could be unambiguously assessed as independent on the basis of specificity, TCR sequences, and Vß^a mouse of origin. The nucleotide and deduced amino acid sequences for the TCR ß- and -chain junctional regions are shown in Fig. 6. The overall HLA-CW3-selected repertoire in Vß^a mice appeared much more diverse than in Vß^b mice for both the TCR ß- and the -chains. Indeed, in marked contrast to the CTL clones derived from Vß^b mice, Vß^a CTL clones did not exclusively use the Vß10 segment, as the expression of Vß1, Vß2, Vß4, and Vß15 was also detected. The CDR3ß sequences of clones expressing the Vß10 segment were different at the nucleotide level from all sequences found in Vß^b CTL clones or those defined by single-cell PCR (11, 12). However, the Vß10⁺ clones 1.2.B1 and 1.2.D2, which recognized the CW3_170–179 peptide, displayed the same ß-chain sequence at the amino acid level as Vß^b clones CW3/2C1 and CW3/A8, respectively. The clone 1.2.B1 also used an -chain that was identical at the amino acid level with a Vß^b CTL clone (CW3/HLA1G6) even though this particular -ß association was not found in any Vß^b CTL clone, whereas the -chain of the clone 1.2.D2 displayed a V segment, a J segment, and a CDR3 sequence completely different from all Vß^b CTL clones. Interestingly, the CDR3ß sequence of the Vß10⁺ clone 1.1.C7 displayed only one nucleotide difference with the sequence 6c (12) described in Vß^b mice (CCC instead of TCC, respectively), which resulted in a Pro residue instead of a Ser residue in position 4 of the CDR3. Collectively, the conserved features of the TCR ß-chain in CW3-specific Vß^b TCRs were not shared by Vß^a TCRs, including Vß usage, a length of 6 aa, and a Gly residue at position 3 of the CDR3ß. In contrast, the clones that did not recognize the CW3_170–179 peptide shared common features in the CDR3ß, including a conserved length of 10 aa and a negatively charged or polar residue at position 3 together with polar residues at positions 4 and 8. The broad Vß usage observed in these CTL clones is in agreement with the absence of preferential Vß usage observed in HLA-CW3 immune Vß^a mice (Fig. 3).

The J usage was likewise more diverse in Vß^a mice, because the CTL clones analyzed in this study used seven different J segments, rearranged in some cases to V segments that were not observed in Vß^b clones (e.g., V10, V12, and V17.1). Furthermore, contrary to Vß^b clones, CDR3 length was broadly distributed in Vß^a TCRs, ranging from 5 to 11 aa. The -chains of the Vß10^a+ clones 1.2.B1 and 1.1.C7 were identical at the amino acid level with Vß^b clones CW3/HLA1G6 and CW3/HLA1C8, with one or two silent differences at the nucleotide level in the CDR3, respectively. Interestingly, the clone 1.1.F3, which does not recognize the CW3_170–179 peptide, also shared -chain junctional sequence with a CW3 peptide-specific Vß^b clone (CW3/701.1); the CDR3 sequence at the amino acid level and J usage were identical, even though they expressed different V segments (V12 for clone 1.1.F3 vs V3.5 for clone CW3/701.1). Finally, similarly to ß-chains, the -chains of Vß^a clones specific for the CW3_170–179 peptide were diverse and did not display the conserved features found in all Vß^b clones (such as J usage and a conserved length of 9 aa), whereas CTL clones that did not recognize the CW3_170–179 peptide displayed structurally similar CDR3 despite a broad V and J usage.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Importance of CDR1ß-peptide interactions for TCR-ligand recognition

Because of its exceptional capacity for combinatorial diversity, the (D)J-encoded CDR3 of the TCR - and ß-chains was initially proposed to be entirely responsible for the specificity of TCR binding to antigenic peptides. According to this model (2), germline V gene-encoded CDR1 and CDR2 would only be responsible for making contacts between the TCR and polymorphic residues present on MHC class I or II molecules. Recent crystallographic analyses of several TCR/peptide/MHC class I trimolecular complexes have nevertheless revealed that the CDR1ß (in addition to the CDR3 and CDR3ß) can make direct contacts with the antigenic peptide (3, 4, 5, 21). To investigate the putative contribution of CDR1ß-peptide contacts to the overall avidity of TCR-ligand interactions in a physiological setting in vivo, we have taken advantage of a naturally occurring CDR1 coding sequence polymorphism in the Vß10 gene segment between the Vß^a and Vß^b haplotypes of mice. Using a well-characterized model system in which CD8⁺ Vß10⁺ T cells dominate the H-2K^d-restricted response to the HLA-CW3 peptide 170–179 in DBA/2 mice, we demonstrate here that CDR1ß polymorphism can have a dramatic impact on the outcome of an MHC class I-restricted T cell response in vivo. Furthermore, because MHC class I-restricted CD8⁺ T cell responses to immunodominant viral epitopes are frequently characterized by restricted Vß usage in several species (36), our data raise the possibility that CDR1ß-peptide interactions are in general required to achieve sufficient TCR-ligand affinity for the triggering of CD8⁺ T cell responses to physiological Ags in vivo. The structural basis of the impact of Vß10 gene polymorphism on the CW3-specific CD8⁺ response in Vß^a mice, and particularly the central role of putative CDR1ß-peptide interactions, are discussed below.

Amino acid changes in Vß10^a are unlikely to modify the overall structure of the TCR

In all Vß^a CTL clones, TCRs using the Vß10^a segment are normally expressed on the cell surface and are efficiently stimulated by anti-CD3 mAbs (data not shown). The fact that we could derive Vß^a CTL clones using the Vß10^a gene segment and recognizing the H-2K^d-CW3 peptide complex suggests that mutations in the Vß10^a gene segment do not alter the conformation of Ag-binding loops. Furthermore, as demonstrated by Vß^a CTL clones 1.1.C7 and 1.2.B1, the Vß10^a segment is able to pair with an -chain that is identical (at the amino acid level) with an -chain used in association with Vß10^b in Vß^b mice. Finally, Gln residues at positions 37 and 25 of the ß-chain, which have been suggested to play an important role in the formation of contact between V and Vß domains and in the stabilization of the CDR1ß loop conformation (4), respectively, are conserved in the Vß10 gene between the Vß^a and Vß^b haplotypes. This observation is in agreement with normal loop conformation and -chain pairing in TCRs using the Vß10^a gene segment.

Interaction with the H-2K^d molecule itself would not be profoundly altered by Vß10 polymorphism

The crystal structure of trimolecular complexes as well as the energy map of the interaction strongly suggested that several residues of the CDR1ß loop were involved in direct contacts with the peptide-MHC complex (3, 5, 37). The crystal structure of the 2C TCR bound to its ligand dEV8-H-2-K^b revealed that residues 26, 28, 29, and 30 in the CDR1ß were contacting conserved residues of the MHC molecule (5). Among these CDR1ß amino acids, residues 28 and 30 are polymorphic between the Vß^a and Vß^b haplotypes. However, Thr²⁶ and above all His²⁹, which are present in both Vß10 alleles, are highly conserved residues in Vß genes, and His²⁹ is thought to play a major role, in combination with several -chain residues, in dictating the general orientation of the TCR toward the peptide-MHC complex and in establishing crucial contact with the MHC molecule (5). Thus, conservation between the Vß10^a and Vß10^b alleles of His²⁹ in the CDR1ß loop and identity in the CDR2ß, which is thought to make multiple contacts with the MHC molecule (5, 21, 37), as well as possible pairing with identical -chains in Vß^a and Vß^b haplotypes, could account for the ability of TCRs expressing Vß10^a to efficiently recognize the peptides presented by H-2-K^d molecules.

Vß10 CDR1 polymorphic residues would establish critical contacts with the C-terminal part of the CW3 peptide

The crystal structure of the trimolecular complexes TCR/dEV8/H-2-K^b and TCR/Tax/HLA-A2 agreed on direct contacts between the C-terminal part of the peptide and both CDR1ß and CDR3ß, whereas CDR1 and CDR3 interact with the N-terminal part of the peptide (3, 4, 5, 21). To evaluate the putative interaction of Vß10 CDR1 with the CW3 peptide, we built a model of the Vß^b 1C8 TCR/CW3 (170–179)/K^d complex based on the three-dimensional crystallographic coordinates of A6 and B7 TCR/Tax/HLA-A2 and 2C TCR/dEV8/H-2K^b complexes (3, 4, 5). According to our model, the three amino acid changes at positions 11, 14, and 84 between the Vß^b and Vß^a haplotypes would not modify the overall TCR structure or the interaction of the TCR with the H-2K^d-CW3 complex (data not shown). By contrast, the diagonal orientation of the TCR relative to the H-2K^d-CW3 complex resulted in the positioning of the Vß10 CDR1 loop above the C-terminal part of the CW3 peptide (Fig. 7). Most importantly, TCR ß-chain residues Gly²⁸ and Asp³⁰ form, respectively, van der Waals contacts and two hydrogen bonds with CW3 peptide residues Glu⁸ and Lys⁷, which appear to be the main residues pointing toward the TCR in the C-terminal part of the CW3 peptide bound to H-2K^d. These two TCR residues are both located in the CDR1ß and are polymorphic between the Vß^b and Vß^a haplotypes (Fig. 1). Introduction into our model of the amino acid changes Gly-Asp (Vß^b-Vß^a) at position 28, Asp-Asn at position 30, and Lys-Glu at position 24 substantially impaired the interaction between the TCR CDR1ß and the C-terminal part of the CW3 peptide mainly by the charge-charge repulsion occurring between Asp²⁸ of the Vß10^a CDR1 and Glu⁸ of the CW3 peptide. Our model is strengthened by a previous analysis of recognition by the CW3-specific Vß10^b+ CTL clones of the CW3 peptide mutated at individual residues, which showed that Ala substitutions at position Lys⁷ and Glu⁸ greatly reduced the relative antigenic activity of the CW3 peptide for all of the clones tested (26). Furthermore, our model is in agreement with reports on the impact of point mutations in the CDR1ß on several Ag-specific responses in vitro (class I- and class II-restricted) (25, 38, 39, 40).

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Modeling of the 1C8 TCR/CW3/H-2Kd complex. The molecular model of the 1C8 TCR/CW3/H-2Kd complex was built based on available TCR/peptide/MHC crystal structures as described in Materials and Methods. This figure presents a stereo view focused on the Vß10 CDR1 (top) and the carboxy-terminal part of the CW3170–179 peptide (bottom) presented by H-2Kd. The dotted lines indicate hydrogen bonds.

HLA-CW3-specific repertoires qualitatively and quantitatively differ in Vß^b and Vß^a mice

Vß^a TCR ß- and -chain junctional sequences revealed interesting features in comparison with Vß^b TCR sequences: Vß^a CTL clones specific for the CW3_170–179 peptide displayed a much broader repertoire than Vß^b CTL clones in terms of Vß usage, J usage, and junctional diversity characterized by diverse CDR3ß and CDR3 lengths and the absence of conserved residues (Fig. 6). Increased diversity in the anti-CW3 Vß^a repertoire could be secondary to mutations in the CDR1ß of the Vß10 gene, which may not allow TCRs bearing the conserved features described in Vß^b mice to be efficiently selected, such that selected TCRs bear diverse Ag-binding loops (in - and ß-chains) that conceivably compensate mutations in the CDR1ß for interaction with the H-2K^d-CW3 complex. In this respect, the correlation found by Pannetier et al. between CDR3ß size and the amino acid sequence in the CDR1ß (41) could (at least partially) explain the diversity of CDR3ß length observed in Vß^a CTL clones. Nevertheless, our results do not allow us to determine whether mutations in the Vß10 CDR1ß increased the affinity of Vß^a TCRs for self peptide-MHC complexes in the thymus, resulting in their negative selection, or conversely whether affinity was decreased below the threshold level required for positive selection.

It has been estimated that only 10–30 clones would compose the anti-CW3 CD8 response in Vß^b mice (12, 42). An additional limitation of the anti-HLA-CW3-specific repertoire secondary to Vß10 polymorphism in Vß^a mice could thus decrease the response below detection levels in most mice and, due to individual variations, even prevent the rejection of P815-CW3 tumor cells in some mice. As shown by H-2K^d-CW3 tetramer staining, the lack of Vß10^a+ cell expansion correlates with the absence of T cells specific for the CW3_170–179 peptide in most Vß^a mice (Fig. 5). Thus, as the anti-CW3 response can be achieved by <30 specific clones, this result implies that T cell precursors specific for the CW3_170–179 peptide are either absent or present at very low number in most Vß^a mice. Conversely, the expansion of CW3-specific Vß10^a+ T cells in several individual Vß^a mice can be explained by diversity in the naive repertoire, as was shown recently in the anti-HLA-CW3 and anti-HLA-A2 repertoires of DBA/2 mice (18). It is noteworthy that in four of five Vß^a mice in which expansion of Vß10^a+ cells could be detected (Figs. 2 and 5), the percentage of Vß10^a+ cells in the activated CD8⁺ T cell population did not reach the level observed in Vß^b mice (30–40% in Vß^a mice compared with 70–80% in Vß^b mice). This result is paralleled by the significantly weaker Vß10^b+ T cell expansion in (Vß^b x Vß^a)F₁ mice compared with Vß^b mice, which could be explained by the presence in F₁ mice of only one copy of the Vß10^b gene segment. Taken together, these results suggest that a very low number of T cell precursors specific for the CW3_170–179 peptide is enough to mount an efficient anti-CW3 response, further suggesting a potential absence of precursors specific for the CW3_170–179 peptide in most Vß^a mice. Thus, the rejection of P815-CW3 tumor cells would be secondary to the recognition of the subdominant or cryptic epitope(s) of the HLA-CW3 molecule in most Vß^a mice. Furthermore, we could not detect any preferential Vß usage associated with this response (Fig. 3), suggesting that the repertoire specific for these novel HLA-CW3 epitope(s) is probably very limited; this possibility would explain why 5 of 29 Vß^a mice failed to reject P815-CW3 tumor cells due to individual variations in the naive repertoire.

General conclusion

To our knowledge, this study is the first direct demonstration of the influence of Vß gene polymorphism on an Ag-specific CD8⁺ T cell response in vivo. We propose that the effect of Vß10 polymorphism on the anti-HLA-CW3 CD8⁺ T cell response in Vß^a mice is principally due to a modification of interactions between the CDR1ß and the C-terminal part of the CW3 peptide. In more general terms, TCR Vß gene polymorphism may be expected to have potential functional consequences on Ag-specific T cell responses and thus should be taken into consideration in the design of peptide-based cancer vaccine strategies.

	Acknowledgments

 
We thank A. Livingstone and K. Tomonari for generously providing the DBA/2 Vß^a mice and KT10a mAb, respectively. We also thank W. Held, D. Hudrisier, and P. Romero for helpful discussions and critical reading of the manuscript and P. Pagé for assistance with the sequencing.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. H.R. MacDonald, Ludwig Institute for Cancer Research, Chemin Des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:

² Abbreviations used in this paper: CDR, complementarity-determining region; P815-CW3, P815 cells transfected with the HLA-CW3 gene; CD62L, CD62 ligand; PE, phycoerythrin; i.d., intradermal(ly).

Received for publication November 20, 1998. Accepted for publication January 14, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Chothia, C., D. R. Boswell, A. M. Lesk. 1988. The outline structure of the T-cell ß receptor. EMBO J. 7:3745.[Medline]
2. Davis, M. M., P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395.[Medline]
3. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide, and HLA-A2. Nature 384:134.[Medline]
4. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An ß T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274:209.[Abstract/Free Full Text]
5. Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279:1166.[Abstract/Free Full Text]
6. Arden, B., S. P. Clark, D. Kabelitz, T. W. Mak. 1995. Mouse T-cell receptor variable gene segment families. Immunogenetics 42:501.[Medline]
7. Wilson, R. K., E. Lai, P. Concannon, R. K. Barth, L. E. Hood. 1988. Structure, organization, and polymorphism of murine and human T-cell receptor and ß chain gene families. Immunol. Rev. 101:149.[Medline]
8. MacDonald, H. R., J.-L. Casanova, J. L. Maryanski, J.-C. Cerottini. 1993. Oligoclonal expansion of major histocompatibility complex class I-restricted cytolytic T lymphocytes during a primary immune response in vivo: direct monitoring by flow cytometry and polymerase chain reaction. J. Exp. Med. 177:1487.[Abstract/Free Full Text]
9. Bour, H., C. Horvath, C. Lurquin, J.-C. Cerottini, H. R. MacDonald. 1998. Differential requirement for CD4 help in the development of an antigen-specific CD8⁺ T cell response depending on the route of immunization. J. Immunol. 160:5522.[Abstract/Free Full Text]
10. Walker, P. R., T. Ohteki, J. A. Lopez, H. R. MacDonald, J. L. Maryanski. 1995. Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response. J. Immunol. 155:3443.[Abstract]
11. Casanova, J.-L., J.-C. Cerottini, M. Matthes, A. Necker, H. Gournier, C. Barra, C. Widmann, H. R. MacDonald, F. Lemonnier, B. Malissen, et al 1992. H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity. J. Exp. Med. 176:439.[Abstract/Free Full Text]
12. Maryanski, J. L., C. V. Jongeneel, P. Bucher, J.-L. Casanova, P. R. Walker. 1996. Single-cell PCR analysis of TCR repertoires selected by antigen in vivo: a high magnitude CD8 response is comprised of very few clones. Immunity 4:47.[Medline]
13. Smith, L. R., A. Plaza, P. A. Singer, A. N. Theofilopoulos. 1990. Coding sequence polymorphisms among Vß T cell receptor genes. J. Immunol. 144:3234.[Abstract]
14. Maryanski, J. L., R. S. Accolla, B. Jordan. 1986. H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells. J. Immunol. 136:4340.[Abstract]
15. Ohteki, T., H. R. MacDonald. 1994. Major histocompatibility complex class I-related molecules control the development of CD4⁺8^- and CD4^-8^- subsets of natural killer 1.1⁺ T cell receptor-/ß⁺ cells in the liver of mice. J. Exp. Med. 180:699.[Abstract/Free Full Text]
16. Brunner, K. T., H. R. MacDonald, J.-C. Cerottini. 1980. Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors: analysis of the clonal progeny of CTL precursors stimulated in vitro with syngeneic tumor cells. J. Immunol. 124:1627.[Medline]
17. Tomonari, K., O. A. Rosenwasser, S. P. Fairchild. 1997. An antibody specific for Tcrb-V10a and Tcrb-V10c. Immunogenetics 46:529.[Medline]
18. Bousso, P., A. Casrouge, J. D. Altman, M. Haury, J. Kanellopoulos, J. P. Abastado, P. Kourilsky. 1998. Individual variations in the murine T cell response to a specific peptide reflect variability in naive repertoires. Immunity 9:169.[Medline]
19. Pala, P., G. Corradin, T. Strachan, R. Sodoyer, B. R. Jordan, J.-C. Cerottini, J. L. Maryanski. 1988. Mapping of HLA epitopes recognized by H-2-restricted cytotoxic T lymphocytes specific for HLA using recombinant genes and synthetic peptides. J. Immunol. 140:871.[Abstract]
20. Casanova, J. L., P. Romero, C. Widmann, P. Kourilsky, J. L. Maryanski. 1991. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J. Exp. Med. 174:1371.[Abstract/Free Full Text]
21. Ding, Y. H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8:403.[Medline]
22. Fremont, D. H., M. Matsumura, E. A. Stura, P. A. Peterson, I. A. Wilson. 1992. Crystal structures of two viral peptides in complex with murine MHC class I H-2K^b. Science 257:919.[Abstract/Free Full Text]
23. Gotoh, O.. 1982. An improved algorithm for matching biological sequences. J. Mol. Biol. 162:705.[Medline]
24. Sali, A., T. L. Blundell. 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234:779.[Medline]
25. Lone, Y. C., M. Bellio, A. Prochnicka-Chalufour, D. M. Ojcius, N. Boissel, T. H. Ottenhoff, R. D. Klausner, J. P. Abastado, P. Kourilsky. 1994. Role of the CDR1 region of the TCR ß chain in the binding to purified MHC-peptide complex. Int. Immunol. 6:1561.[Abstract/Free Full Text]
26. Maryanski, J. L., J.-L. Casanova, K. Falk, H. Gournier, C. Jaulin, P. Kourilsky, F. A. Lemonnier, R. Luthy, H. G. Rammensee, O. Rotzschke, C. Servis, J. A. Lopez. 1997. The diversity of antigen-specific TCR repertoires reflects the relative complexity of epitopes recognized. Hum. Immunol. 54:117.[Medline]
27. Brooks, B. R., R. E. Bruccoleri, B. D. Olafson, D. J. States, M. Karplus. 1983. CHARMM: a program for macromolecular energy, minimization, and dynamics calculation. J. Comp. Chem. 4:187.
28. Mackerell, A. D., D. Bashford, M. Bellott, R. L. Dunbrack, J. D. Evanseck, M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, et al 1998. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102:3586.
29. Behlke, M. A., H. S. Chou, K. Huppi, D. Y. Loh. 1986. Murine T-cell receptor mutants with deletions of ß-chain variable region genes. Proc. Natl. Acad. Sci. USA 83:767.[Abstract/Free Full Text]
30. Luther, S. A., H. Acha-Orbea. 1997. Mouse mammary tumor virus: immunological interplays between virus and host. Adv. Immunol. 65:139.[Medline]
31. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⁺ T cells during an acute virus infection. Immunity 8:167.[Medline]
32. Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8⁺ T cells during the primary immune response to Epstein-Barr virus in vivo. J. Exp. Med. 187:1395.[Abstract/Free Full Text]
33. Gallimore, A., A. Glithero, A. Godkin, A. C. Tissot, A. Pluckthun, T. Elliott, H. Hengartner, R. Zinkernagel. 1998. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187:1383.[Abstract/Free Full Text]
34. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
35. Crawford, F., H. Kozono, J. White, P. Marrack, J. Kappler. 1998. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8:675.[Medline]
36. Casanova, J.-L., J. L. Maryanski. 1993. Antigen-selected T-cell receptor diversity and self-nonself homology. Immunol. Today 14:391.[Medline]
37. Manning, T. C., C. J. Schlueter, T. C. Brodnicki, E. A. Parke, J. A. Speir, K. C. Garcia, L. Teyton, I. A. Wilson, D. M. Kranz. 1998. Alanine scanning mutagenesis of an ß T cell receptor: mapping the energy of antigen recognition. Immunity 8:413.[Medline]
38. Bellio, M., Y. C. Lone, O. de la Calle-Martin, B. Malissen, J. P. Abastado, P. Kourilsky. 1994. The Vß complementarity-determining region 1 of a major histocompatibility complex (MHC) class I-restricted T cell receptor is involved in the recognition of peptide/MHC I and superantigen/MHC II complex. J. Exp. Med. 179:1087.[Abstract/Free Full Text]
39. Gahm, S. J., B. J. Fowlkes, S. C. Jameson, N. R. Gascoigne, M. M. Cotterman, O. Kanagawa, R. H. Schwartz, L. A. Matis. 1991. Profound alteration in an ß T-cell antigen receptor repertoire due to polymorphism in the first complementarity-determining region of the ß chain. Proc. Natl. Acad. Sci. USA 88:10267.[Abstract/Free Full Text]
40. Kasibhatla, S., E. A. Nalefski, A. Rao. 1993. Simultaneous involvement of all six predicted antigen-binding loops of the T cell receptor in recognition of the MHC/antigenic peptide complex. J. Immunol. 151:3140.[Abstract]
41. Pannetier, C., M. Cochet, S. Darche, A. Casrouge, M. Zöller, P. Kourilsky. 1993. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor ß chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA 90:4319.[Abstract/Free Full Text]
42. Walker, P. R., A. Wilson, P. Bucher, J. L. Maryanski. 1996. Memory TCR repertoires analyzed long-term reflect those selected during the primary response. Int. Immunol. 8:1131.[Abstract/Free Full Text]
43. Chien, Y. H., R. J. Gascoigne, J. Kavaler, N. E. Lee, M. M. Davis. 1984. Somatic recombination in a murine T-cell receptor gene. Nature 309:322.[Medline]
44. Gascoigne, N. R., Y. Chien, D. M. Becker, J. Kavaler, M. M. Davis. 1984. Genomic organization and sequence of T-cell receptor ß-chain constant- and joining-region genes. Nature 310:387.[Medline]
45. Koop, B. F., L. Rowen, K. Wang, C. L. Kuo, D. Seto, J. A. Lenstra, S. Howard, W. Shan, P. Deshpande, L. Hood. 1994. The human T-cell receptor TCRAC/TCRDC (C/C) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19:478.[Medline]

This article has been cited by other articles:

				P. Brawand, J.-C. Cerottini, and H. R. MacDonald Hierarchal Utilization of Different T-Cell Receptor Vbeta Gene Segments in the CD8+-T-Cell Response to an Immunodominant Moloney Leukemia Virus-Encoded Epitope In Vivo J. Virol., November 1, 1999; 73(11): 9161 - 9169. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bour, H. Articles by MacDonald, H. R. Search for Related Content PubMed PubMed Citation Articles by Bour, H. Articles by MacDonald, H. R.