Hierarchal Utilization of Different T-Cell Receptor Vβ Gene Segments in the CD8⁺-T-Cell Response to an Immunodominant Moloney Leukemia Virus-Encoded Epitope In Vivo

Abstract

The CD8⁺-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D^b and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Vα3.2 and Vβ5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vβ^a mice which are unable to express the dominant Vα3.2⁺ Vβ5.2⁺ T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vβ5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vβ^a mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8⁺ cells utilizing the same Vα3.2 gene segment in association with two different Vβ segments (Vβ3 and Vβ17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 × C57BL/6 Vβ^a)F₁ mice revealed a clear hierarchy in Vβ utilization, with a preferential usage of the Vβ17 gene segment, whereas Vβ3 and especially Vβ5.2 were used to much lesser extents. Sequencing of TCRα- and -β-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRβ chains in Vβ^a mice and a highly restricted TCRβ-chain repertoire in Vβ^b mice, whereas TCRα-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D^b-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vβ domains and that the degree of β-chain diversity varies according to Vβ utilization.

Cytotoxic T lymphocytes (CTL) play an important role in the eradication of intracellular pathogens. CTL become activated when their T-cell receptors (TCR) specifically recognize foreign antigen in the form of a molecular complex between a major histocompatibility complex (MHC) class I molecule and a short antigenic peptide (31, 33). TCR molecules are heterodimers whose α and β chains are composed of a constant (C) and a variable (V) extracellular domain. Somatic recombination of a number of V, diversity (D), and junctional (J) gene segments, imprecise joining, and addition of N nucleotides are responsible for the TCRα- and -β-chain diversity which is further increased by combinatorial α-β pairing (38). The specificity of the TCR is determined mainly by three hypervariable complementarity-determining regions (CDR) of the α and β chains. CDR1 and CDR2 are encoded by V segments, whereas CDR3 is encoded by J elements for the α chain and by D and J elements for the β chain. This exceptional potential diversity (estimated at more than 10¹⁵ for TCR) allows CD8 T cells to respond to a wide variety of antigens (14).

Recent studies have focused on the diversity of TCR in recognizing one given antigen (9). For example, TCR expressed by CTL clones specific for a single Plasmodium berghei circumsporozoite peptide were highly diverse in terms of Vα, Jα, and Jβ segments and amino acid compositions of the junctional regions. However the Vβ segment utilization was strongly conserved among the different clones (10). In contrast, analysis of the TCR repertoire of CTL directed against a peptide derived from the human class I MHC molecule HLA-CW3 presented by murine MHC class I (H-2K^d) molecules at the surface of P815 tumor cells revealed a very limited heterogeneity both in terms of Vα, Jα, Vβ, and Jβ segments and in terms of lengths and sequences of both CDR3α and -β (8). This highly restricted TCR usage allowed the identification of antigen-specific T cells in individual immune DBA/2 mice either by staining with monoclonal antibodies (MAbs) to the Vβ domain (25) or by single-cell PCR (26). In addition, Vβ preferences have been demonstrated in CD8⁺ T-cell responses to acute infection by several viruses, including human immunodeficiency virus (30), simian immunodeficiency virus (12), Epstein-Barr virus (7) and lymphocytic choriomeningitis virus (24).

In a previous publication (5), we showed that the CD8⁺ T cells responsible for the rejection of Moloney murine leukemia virus (M-MuLV)-induced tumor cells had a very restricted usage of both Vα and Vβ gene segments. Indeed, immunization of C57BL/6 (B6) mice with M-MuLV-induced tumor cells (MBL-2) led to an overwhelming expansion of CD8⁺ T cells that recognized exclusively a virally encoded immunodominant epitope. This epitope (CCLCLTVFL), presented by H-2D^b, is shared by leukemia and lymphoma cell lines infected by the Friend-Moloney-Rauscher (FMR) group of leukemia viruses and is encoded in the leader sequence of the gag polypeptide (11, 22). These M-MuLV gag-specific CD8⁺ T cells could be readily monitored ex vivo by flow cytometry since the majority coexpressed the Vα3.2 and Vβ5.2 gene segments.

In the present study, we were interested in analyzing the CD8⁺-T-cell response to M-MuLV-induced tumor cells in mice unable to express the dominant Vα3.2⁺ Vβ5.2⁺ TCR. For this purpose we took advantage of congenic B6.Vβ^a mice (28) that have a large deletion at the TCRβ locus, including the Vβ5.2 gene segment (2). Interestingly, despite the absence of Vβ5.2⁺ cells, B6.Vβ^a mice were able to reject M-MuLV-induced tumor cells. Moreover, analysis of the TCR repertoire in these immune B6.Vβ^a mice indicated a dramatic expansion of CD8⁺ T cells coexpressing Vα3.2 together with either the Vβ3 or the Vβ17 gene segment and recognizing the same immunodominant gag epitope. Interestingly, these two Vβ gene segments differ between the Vβ^a and Vβ^b haplotypes. Indeed, the Vβ17 gene is not expressed in the Vβ^b haplotype due to the presence of a stop codon whereas the Vβ3 gene segment differs between the two haplotypes by a point mutation, resulting in a single amino acid substitution at position 31 (Phe in Vβ^a versus Val in Vβ^b) (32). In (B6 × B6.Vβ^a)F₁ mice immunized with M-MuLV-induced tumor cells, we observed a clear hierarchy in Vβ usage by CD8⁺ T cells (Vβ17 > Vβ3 > Vβ5.2). The structural basis for the hierarchal recognition of a single peptide-MHC complex by TCR utilizing three distinct Vβ domains was also investigated by sequencing the CDR3 regions of both the α and β chains.

MATERIALS AND METHODS

Mice.

B6 mice were obtained from HARLAN OLAC (Bicester, United Kingdom). Congenic B6.Vβ^a mice were kindly provided by A. Livingstone (Basel Institute for Immunology, Basel, Switzerland). These mice were derived by transferring the Vβ^a haplotype (which has an extensive deletion at the TCRβ locus, including the Vβ5, -8, -9, -11, -12, and -13 gene segments [2]) from C57L mice (H-2^b, Vβ^a) to B6 mice (H-2^b, Vβ^b). However, two other Vβ gene segments (Vβ17 and Vβ19) that are not expressed in Vβ^b mice are expressed in the Vβ^a haplotype. The B6.Vβ^a mice used were backcrossed for 15 generations to B6 mice. (B6 × B6.Vβ^a)F₁ mice were bred in our animal facilities.

Immunizations.

M-MuLV-infected MBL-2 (H-2^b) tumor cells were maintained by weekly passage in syngeneic B6 mice (6). For primary immunization, 40 × 10⁶ irradiated (10,000 rads) tumor cells were injected intraperitoneally into syngeneic mice. After 3 to 4 weeks, secondary responses were elicited by intraperitoneal injection of 10 × 10⁶ viable syngeneic tumor cells.

MLTC and CTL clones.

Virus-specific CTL were generated in vitro in a 5-day mixed lymphocyte-tumor cell culture (MLTC) (3). Responder spleen cells (25 × 10⁶) from M-MuLV immune mice and irradiated MBL-2 cells (1 × 10⁶) were cocultured in 15 ml of Dulbecco modified Eagle medium (Gibco, Paisley, United Kingdom) supplemented with 2 × 10⁻³ M l-glutamine, 2 × 10⁻² M HEPES, 3 × 10⁻⁵ M 2-mercaptoethanol, antibiotics, and 5% heat-inactivated fetal calf serum (Irvine Scientific, Santa Ana, Calif.). Cells recovered from MLTC were washed and restimulated with irradiated MBL-2 and syngeneic feeder cells for a further seven days in complete medium supplemented with 30 U of interleukin 2 (EL-4 cell supernatant) per ml. CTL clones were established by plating cells from an MLTC at limiting dilution as described previously (5).

Cytotoxic assays.

CTL clones derived from M-MuLV-immune mice were used as effector cells. Target cells were either MBL-2 lymphoma (M-MuLV infected, H-2^b), RMA lymphoma (Rauscher virus infected, H-2^b), or EL-4 lymphoma (FMR uninfected, H-2^b) cells. The FMR gag-encoded epitope CCLCLTVFL (11) was synthesized and purified by standard procedures and dissolved in dimethyl sulfoxide supplemented with β-mercaptoethanol. For cytotoxic assays, effector cells and ⁵¹Cr-labeled target cells were mixed at the ratios indicated below in the presence or absence of various concentrations of peptide. Supernatants were harvested after 4 h, and specific ⁵¹Cr release was calculated as described previously (5).

Flow microfluorometry.

At various times after primary or secondary immunization with syngeneic M-MuLV-infected tumor cells, mice were bled by the tail vein and peripheral blood lymphocytes (PBL) were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden).

Four-color analyses of isolated PBL from M-MuLV-immune mice were performed with MAbs to CD8 (53-6.7), CD4 (RM4-5), and CD62L (Mel-14) and a panel of anti-Vβ MAbs including Vβ2 (B20.6), Vβ3 (KJ25), Vβ4 (KT4), Vβ5 (MR9-4), Vβ6 (44-22), Vβ7 (TR310), Vβ14 (14-2), and Vβ17 (KJ23) in conjunction with a panel of anti-Vα MAbs including Vα2 (B20.1), Vα3.2 (RR3-16), Vα8 (B21.14), and Vα11 (RR8-1). CTL clones or MLTCs were triple stained with MAbs to CD8, Vβ, and Vα. All samples were gated on viable cells (assessed by light scatter) and run on either a FACSCalibur or a FACStar (Becton Dickinson, San Jose, Calif.) equipped with either CellQuest or LYSIS II software, respectively.

RNA extraction, cDNA synthesis, and PCR.

Total RNA was extracted from 5 × 10⁶ cells from MLTCs or CTL clones with QIAshredder columns as the cell lyzate homogenizer and an RNeasy Mini Kit as the RNA extraction system (both from Qiagen AG, Basel, Switzerland). Single-stranded cDNA synthesis was carried out on total RNA with oligo(dT)15 and avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). PCR was carried out in 50 μl on 2/50 of the cDNA with 5 U of Taq polymerase (Eurobio) according to the manufacturer’s instructions. Oligonucleotides for the PCR amplification were the following: (Vα3) 5′-AAGTACTATTCCGGAGACCC-3′, (Cαa) 5′-TGGCGTTGGTCTCTTTGAAG-3′, (Cαb) 5′-ACACAGCAGGTTCTGGGTTC-3′, (Vβ3) 5′-CCTTGCAGCCTAGAAATTCAGTCC-3′, (Vβ5.2) 5′-AAGGTGGAGAGAGACAAAGGATTC-3′, (Vβ17) 5′-GAACAAACAGACTTGGTCAAG-3′, (Cβa) 5′-CCAGAAGGTAGCAGAGACCC-3′, and (Cβb) 5′-CTTGGGTGGAGTCACATTTCTC-3′ (10). Forty cycles, each of 94°C for 15 s, 58°C for 45 s, and 72°C for 60 s, were completed in a thermocycler. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen AG).

Sequencing reactions.

Sequencing reactions of purified PCR products were done by fluorescent cycle sequencing with a Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Life Science Ltd.) according to the manufacturer’s instructions and analyzed in a LI-COR DNA sequencer (MWG-biotech, Munchenstein, Switzerland).

RESULTS

TCR Vα and Vβ repertoires of M-MuLV-immune B6.Vβ^a mice.

B6.Vβ^a mice were injected with irradiated syngeneic M-MuLV-infected (MBL-2) tumor cells and boosted 2 weeks later with viable cells. Immune mice were able to reject the tumor. These mice were then bled at day 7 after the second injection, and PBL were pooled and stained with a panel of anti-Vα or anti-Vβ MAbs together with anti-CD4 or anti-CD8 MAb. MAbs against CD62L (Mel-14) were included in the fourth color to increase the sensitivity of detection of responding CD8⁺ or CD4⁺ cells and to be able to differentiate between activated (CD62L⁻) and nonactivated (CD62L⁺) T cells (36). The TCR Vα and Vβ repertoires of M-MuLV-immune B6.Vβ^a mice are shown in Fig. 1. PBL from M-MuLV-immune B6.Vβ^a mice were highly enriched for Vα3.2⁺, Vβ3⁺, and Vβ17⁺ cells in the activated (CD62L⁻) subset of CD8⁺ cells following secondary immunization with MBL-2 cells. The other Vα and Vβ domains tested showed lower levels in the CD62L⁻ compartment than in the CD62L⁺ subset. As expected, no preferential TCR Vα or Vβ usage was observed among the nonactivated (CD62L⁺) subset of CD8⁺ cells or among activated (CD62L⁻) CD4⁺ PBL.

FIG. 1.

TCR Vα and Vβ repertoires of M-MuLV-immune PBL in B6.Vβ^a mice. PBL from a pool of 15 B6.Vβ^a mice immunized twice with syngeneic M-MuLV-infected MBL-2 tumor cells were stained in four colors with MAbs to CD8, CD4, and CD62L and a panel of anti-Vα or anti-Vβ MAbs. Open and filled bars represent percentages of CD8⁺ or CD4⁺ cells expressing the indicated Vα or Vβ domain in the CD62L⁺ or CD62L⁻ subsets, respectively.

M-MuLV-immune CD8⁺ cells in B6.Vβ^a mice preferentially express Vα3.2 in association with either Vβ3 or Vβ17.

PBL from 15 M-MuLV immune B6.Vβ^a mice were pooled, and the expression of Vα3.2 versus Vβ3 or Vβ17 was analyzed either among activated (CD62L⁻) or nonactivated (CD62L⁺) CD8⁺ cells (Fig. 2). CD62L⁺ CD8⁺ PBL from immune and naive mice showed the same small percentage of Vα3.2⁺, Vβ3⁺, or Vβ17⁺ cells (data not shown). In contrast, a dramatic expansion of activated CD8⁺ cells expressing Vα3.2 in exclusive association with either Vβ3 or Vβ17 was observed in CD62L⁻ CD8⁺ PBL from M-MuLV-immune mice. The majority of the cells (55%) in this subset were Vα3.2⁺ Vβ17⁺, whereas 11% of the cells were Vα3.2⁺ Vβ3⁺ (Fig. 2B). Further analysis of 26 individual M-MuLV-immune B6.Vβ^a mice revealed a good correlation between the percentage of Vα3.2⁺ and the percentage of Vβ3⁺ and/or Vβ17⁺ cells in the CD62L⁻ CD8⁺ subpopulation (Fig. 3A). Nevertheless the percentages of Vα3.2⁺ and Vβ3⁺ and/or Vβ17⁺ cells in the CD62L⁻ CD8⁺ subset were quite variable among individual immune mice and in a few cases did not exceed backgrounds levels found in normal mice.

FIG. 2.

M-MuLV-immune CD62L⁻ CD8⁺ PBL in B6.Vβ^a mice preferentially express Vα3.2 in association with either Vβ3 or Vβ17. Four-color analyzes of isolated PBL from M-MuLV-immune B6.Vβ^a mice were performed with MAbs to CD8, CD62L, Vα3.2, and Vβ3 or Vβ17. (A) The cytogram represents the staining of CD62L versus CD8. Region 1 (R1) represents activated CD8⁺ (CD62L⁻) cells, whereas region 2 (R2) represents nonactivated CD8⁺ (CD62L⁺) cells. (B) The four cytograms represent Vα3.2 staining versus Vβ3 or Vβ17 expression in the indicated subsets. (C) The three histograms represent Vα3.2, Vβ3, and Vβ17 staining gated on CD62L⁻ CD8⁺ (shaded area) or CD62L⁺ CD8⁺ (nonshaded area) cells.

FIG. 3.

Expression of Vα3.2 in association with either Vβ3 or Vβ17 on CD8⁺ PBL from individual M-MuLV-immune B6.Vβ^a mice. Four-color analyzes of isolated PBL from 39 individual M-MuLV immune B6.Vβ^a mice were performed with MAbs to CD8, CD62L, Vα3.2, and Vβ3 or Vβ17. (A) Correlation curve between the percentages of CD8⁺ CD62L⁻ PBL expressing Vα3.2 in association with either Vβ3 or Vβ17. (B) Open and filled bars indicate, respectively, the percentages of Vβ17⁺ and Vβ3⁺ cells in the CD8⁺ CD62L⁻ subset.

Down-regulation of TCR and CD8 expression in M-MuLV-immune T cells.

In accordance with the observations made for PBL from M-MuLV-immune B6 mice, between 30 and 50% of CD8⁺ cells from M-MuLV-immune B6.Vβ^a mice were CD62L⁻ at the peak of the response whereas only 2 to 3% of total CD8⁺ cells from naive animals had this activated phenotype (data not shown). In addition, a down-regulation of both CD8 and TCR expression was observed in this activated CD62L⁻ subset of CD8 cells. Indeed, the level of expression of CD8, Vα3.2, Vβ3, and Vβ17 was down-regulated by two- to threefold in activated CD8⁺ cells (Fig. 2B and C). This result, which was previously observed in other model systems (5, 36), suggests that down-regulation of both the TCR and coreceptor is a common feature of antigen-specific activation of CD8⁺ cells in vivo.

Hierarchy of Vβ3 and Vβ17 gene usage among CD62L⁻ CD8⁺ cells from individual M-MuLV-immune B6.Vβ^a mice.

Representative data describing the percentages of Vβ3⁺ or Vβ17⁺ cells among the CD62L⁻ CD8⁺ subsets of 39 individual M-MuLV-immune B6.Vβ^a mice are shown in Fig. 3B. The majority of the mice showed a strong expansion of specific CD8⁺ cells expressing Vβ3⁺ and/or Vβ17⁺ gene segments (between 60 and 80% of CD62L⁻ CD8⁺ cells were Vβ3⁺ or Vβ17⁺). The ratio of the percentages of Vβ3⁺ and Vβ17⁺ cells among activated CD8⁺ cells varied from mouse to mouse. In the majority of the responding mice, the Vβ17⁺ response was predominant compared to the Vβ3⁺ response. However, in some mice, Vβ3 and Vβ17 responses were equivalent and even in one mouse, the response was due mainly to Vβ3⁺ cells.

Some mice did not show a preferential expansion of Vβ3⁺ or Vβ17⁺ cells, as was indicated by the fact that the same level of Vβ3⁺ or Vβ17⁺ cells was found in the CD62L⁻ and CD62L⁺ CD8⁺ subsets (Fig. 3B). Interestingly, these immune mice were still able to reject the tumor and showed a significant expansion of CD62L⁻ CD8⁺ cells (data not shown). These results suggest that other TCR molecules can be utilized by B6.Vβ^a mice in order to respond to M-MuLV-infected cells.

Absolute magnitude of the M-MuLV-specific CD8⁺-T-cell response in immune B6.Vβ^a mice.

Since these experiments were performed by analyzing antigen-specific cells via four-color staining, it was possible to calculate the absolute magnitudes of the different subsets of M-MuLV-specific CD8⁺ cells at the peak of the response in immune B6.Vβ^a mice. Vα3.2⁺ Vβ3⁺ CD62L⁻ cells accounted on average for 0.5% of the CD8 subset and 0.05% of total PBL in these mice, whereas the proportions observed in naive animals were, respectively, <0.05% and <0.01%. The absolute number of Vα3.2⁺ Vβ17⁺ CD62L⁻ cells was even higher, since 5% of the CD8 subset and 0.05% of PBL in immune mice had this phenotype compared to <0.1% and <0.01%, respectively, in naive mice. It is important to point out that significant variations from mouse to mouse were observed.

Vα3.2⁺ Vβ3⁺ and Vα3.2⁺ Vβ17⁺ CTL clones predominantly recognize the dominant FMR gag-encoded epitope.

In B6 mice, the protective CD8⁺ CTL response is restricted by the H-2D^b molecule and inhibited by anti-H-2D^b MAb (37). A recent study has shown that the protective CD8⁺ CTL response is directed against an immunodominant epitope (CCLCLTVFL) encoded in the leader sequence of the gag polypeptide of M-MuLV (11). This epitope is shared by leukemia and lymphoma cell lines infected by the FMR group of leukemia viruses.

Since Vα3.2⁺ Vβ3⁺ and Vα3.2⁺ Vβ17⁺ cells show a dramatic expansion among activated CD8⁺ cells from M-MuLV-immune B6.Vβ^a mice, we tested three Vα3.2⁺ Vβ3⁺ and four Vα3.2⁺ Vβ17⁺ CTL clones for their ability to lyse EL-4 lymphoma cells (an H-2^b tumor not infected by FMR retroviruses) in the presence or absence of the CCLCLTVFL peptide and compared the results to those obtained with Vα3.2⁺ Vβ5.2⁺ CTL clones derived from immune B6 mice (5).

The FMR gag peptide was indeed efficient in promoting lysis of EL-4 lymphoma cells by all Vα3.2⁺ Vβ3⁺, Vα3.2⁺ Vβ17⁺, and Vα3.2⁺ Vβ5.2⁺ CTL clones tested in a dose-dependent manner (see representative examples in Fig. 4), indicating that the CCLCLTVFL epitope is recognized in the context of H-2D^b by CD8⁺-T-cell clones bearing the same Vα domain (Vα3.2) in association with at least three different Vβ domains (Vβ3, Vβ5.2, and Vβ17).

FIG. 4.

Vα3.2⁺ Vβ3⁺ and Vα3.2⁺ Vβ17⁺ T-cell clones recognize the dominant gag-encoded epitope. Representative individual M-MuLV-specific CTL clones 6 (Vα3.2⁺, Vβ5.2⁺, H-2D^b-restricted, derived from a B6 mouse), 471 (Vα3.2⁺, Vβ17⁺, H-2D^b restricted, derived from a B6.Vβ^a mouse), 487 (Vα3.2⁺, Vβ3⁺, H-2D^b restricted, derived from a B6.Vβ^a mouse), and 464 (Vα3.2⁻, Vβ5.2⁻, H-2K^b restricted, derived from a B6.Vβ^a mouse) were tested for cytotoxicity at an effector cell/target cell ratio of 3:1 against EL-4 target cells (H-2^b, FMR uninfected) in the presence or absence of various concentrations of the FMR gag-encoded peptide CCLCLTVFL.

Hierarchy of Vβ gene usage in M-MuLV-immune (B6 × B6.Vβ^a)F₁ mice.

In order to further analyze hierarchy in Vβ usage in the M-MuLV immune response, (B6 × B6.Vβ^a)F₁ mice, which express Vβ3, Vβ5.2, and Vβ17 gene segments, were bred in our animal facilities and immunized with MBL-2 tumor cells by the same immunization protocol. PBL from M-MuLV-immune (B6 × B6.Vβ^a)F₁ mice were highly enriched for Vα3.2⁺ cells in the activated (CD62L⁻) subset of CD8⁺ cells following secondary immunization with MBL-2 cells (data not shown). Regarding Vβ usage, we found with 25 individual mice analyzed that activated CD8⁺ cells preferentially utilized the Vβ3 and Vβ17 gene segments and with some mice that they scarcely utilized the Vβ5.2 gene segment (Fig. 5A). In addition, strong individual differences in Vβ3/Vβ17 ratios were observed in MuLV-immune (B6 × B6.Vβ^a)F₁ mice, confirming the observations for B6.Vβ^a mice. Of 25 immune F₁ mice analyzed, most preferentially utilized Vβ17 while one mouse exclusively utilized Vβ3 and others equally utilized the two dominant Vβ chains. A minor expansion of Vβ5.2⁺ cells was also seen in a few mice.

FIG. 5.

Hierarchy of Vβ gene usage in individual M-MuLV-immune (B6 × B6.Vβ^a)F₁ mice. Four-color analyzes of isolated PBL from 25 individual M-MuLV-immune (B6 × B6.Vβ^a)F₁ mice were performed with MAbs to CD8, CD62L, Vα3.2, Vβ3, Vβ5.2, and Vβ17. (A) Open, hatched, and filled bars indicate, respectively, the percentages of Vβ17⁺, Vβ5.2⁺, and Vβ3⁺ cells in the CD8⁺ CD62L⁻ subset. (B) Kinetics of the Vβ17⁺, Vβ5.2⁺, and Vβ3⁺ CD8⁺-T-cell response in three representative M-MuLV-immune (B6 × B6.Vβ^a)F₁ mice.

Longitudinal analyses further demonstrated that the preferential Vβ usage among CD62L⁻ CD8⁺ PBL decreased slowly over time but remained elevated for at least 150 days after immunization (Fig. 5B). However, absolute numbers of Vβ-restricted CD8⁺ cells decreased rapidly starting at day 20 after immunization since the proportion of CD62L⁻ CD8⁺ cells decreased at that time.

TCRα and -β junctional sequences of M-MuLV-specific CTL clones derived from immune B6.Vβ^a and B6 mice.

The recognition of the gag-encoded immunodominant epitope (CCLCLTVFL) by TCR molecules having the same Vα chain (Vα3.2) but in association with at least three different Vβ chains (Vβ3, Vβ5.2, and Vβ17) prompted us to analyze at the molecular level TCRα and -β junctional regions of specific CTL clones derived from immune B6 and B6.Vβ^a mice to see if there was any conserved sequence in these critical regions.

A series of 15 H-2D^b-restricted CTL clones derived from immune B6 or B6.Vβ^a mice plus bulk cultures (MLTC, two restimulations in vitro) derived from two different immune B6 mice were analyzed by reverse transcription-PCR with a sense Vα3 primer in conjunction with an antisense Cα primer. The TCR-Vβ amplification was done with a panel of sense Vβ primers (Vβ3, Vβ5.2, and Vβ17) in conjunction with an antisense Cβ primer. Sequencing reactions were done by using either the same sense Vα or Vβ primers or other internal antisense Cα or Cβ primers located closer to the V(D)JC junction. All CTL clones analyzed were restricted by H-2D^b and specific for the M-MuLV immunodominant epitope (CCLCLTVFL). The clones were divided in two groups: those derived from B6 mice (Vα3.2⁺ Vβ5.2⁺) (Fig. 6A) and those derived from B6.Vβ^a mice (Vα3.2⁺ Vβ3⁺ or Vα3.2⁺ Vβ17⁺) (Fig. 6B).

FIG. 6.

TCRα and -β junctional amino acid sequences of M-MuLV-specific CTL clones derived from immune B6.Vβ^a and B6 mice. Fifteen H-2D^b-restricted CTL clones derived from immune B6 (A) or B6.Vβ^a (B) mice plus bulk cultures (MLTC) derived from two different immune B6 mice are listed on the vertical axis. The Vα, Vβ, Jα, and Jβ segment usages are reported. Nomenclature and sequences for Vβ and Vα segments are as described by Arden et al. (1). Jβ sequences are from the work of Gascoigne et al. (20) and Chien et al. (13), and Jα sequences are from the work of Koop et al. (23). Vα and Vβ usage were confirmed by surface staining with corresponding antibodies. The deduced amino acid sequences (in single-letter code) of the junctional, hypervariable, and putatively CDR3-like regions are indicated. The presumed immunoglobulin-like loop, designated CDR3 for convenience, is supported by two framework branches (FW).

Sequencing of TCR from seven Vα3.2⁺ Vβ5.2⁺ CTL clones revealed a dramatic conservation in both the α and β junctional regions (Fig. 6A). Of 50 Jα gene segments, only one (Jα13) was utilized by all the clones and even by CTL derived from two independent bulk cultures (MLTC I and II). In addition, analysis of the β junctional regions of these CTL clones revealed the utilization of only one Jβ gene segment (Jβ1.4). This restricted junctional usage was highlighted by the fact that both the CDR3α and CDR3β regions were totally conserved among the different clones, with an 8-amino-acid sequence (TPTSGGNY) for CDR3α and a 10-amino-acid sequence (SLVGGGNERL) for CDR3β.

We then made the same analyses of seven B6.Vβ^a-derived CTL clones. TCRα junctional region analysis revealed that again only one Jα was utilized by Vβ3⁺ or Vβ17⁺ clones; however, this segment (Jα6) was different from that used by Vβ5.2⁺ CTL clones (Fig. 6B). The CDR3α regions were in general strongly conserved among these CTL clones. We found a 9-amino-acid CDR3α consensus sequence, with only some differences in the second and third amino acids (SXXSNTNKV). In contrast, TCRβ junctional sequences of B6.Vβ^a mice-derived clones revealed major differences from B6 mice-derived clones. First, Vβ3⁺ or Vβ17⁺ CTL clones utilized three different Jβ gene segments (Jβ1.2, Jβ2.4, and Jβ2.6), and second, the CDR3β region was much less conserved, with different lengths (from 6 to 11 amino acids) and no consensus sequence.

DISCUSSION

The CD8⁺-T-cell response to M-MuLV (and, in general, FMR)-associated antigens in B6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) and is restricted primarily to CTL expressing the Vβ5.2 and Vα3.2 gene segments (5). The rationale of the experiments presented here was to examine the M-MuLV response in congenic B6.Vβ^a mice which are unable to express the dominant Vα3.2⁺ Vβ5.2⁺ TCR due to a large deletion at the TCRβ locus that includes the Vβ5.2 gene segment. Interestingly, B6.Vβ^a mice were still able to reject M-MuLV-infected tumor cells and CTL from these immune mice utilized the same Vα3.2 gene segment in association with two different Vβ segments (Vβ3 and Vβ17). Surprisingly, these CTL recognized the same immunodominant M-MuLV gag epitope.

The fact that the same H-2D^b-restricted gag-encoded peptide CCLCLTVFL is recognized by Vβ3⁺ and Vβ17⁺, as well as Vβ5.2⁺, TCR strengthens the argument that this is indeed a highly immunodominant epitope of M-MuLV (at least in H-2^b mice). This immunodominance is somewhat surprising in view of the fact that the M-MuLV gag peptide lacks a classical H-2D^b anchor residue at position 5. Indeed substitution of the normal H-2D^b anchor residue (Asp) for Leu at this position significantly increases the ability of the gag peptide to bind to H-2D^b. However, recognition of the modified peptide by CTL is greatly diminished (4a). The mechanism underlying the immunodominance of the gag epitope in H-2^b mice remains to be established. In this respect the hydrophobic nature of the peptide (which is encoded in the leader sequence of the gag polyprotein) or its putative ability to be efficiently processed may play a role.

Although CTL clones specific for the immunodominant FMR gag epitope were readily elicited in both B6 and B6.Vβ^a strains, there was considerable variation among individual M-MuLV-immune mice in the frequencies of CD62L⁻ CD8⁺ PBL expressing the Vα3.2 gene segment in association with Vβ3, Vβ5.2, or Vβ17. Nevertheless, all primed mice were able to reject M-MuLV-infected tumor cells. These data suggest that other CTL epitopes (in addition to the immunodominant one) are involved in protective immunity to M-MuLV-induced tumors. Moreover, helper-T-cell epitopes, which have been shown to be important for vaccination against FMR tumors (29), are likely to play an important role.

In interpreting the potential hierarchy of Vβ usage in the M-MuLV response, it is important to note that the Vβ3 and Vβ17 gene segments utilized by most M-MuLV-specific CTL in Vβ^a mice differ between the Vβ^a and Vβ^b haplotypes. In particular, the Vβ17 gene is not expressed in the Vβ^b haplotype due to the presence of a stop codon. Moreover, the Vβ3 gene segment differs between the two haplotypes by a point mutation, resulting in a single amino acid substitution at position 31 (Phe in Vβ^a versus Val in Vβ^b). This polymorphic residue is located within the CDR1β domain of the Vβ3 segment and thus might be expected to influence TCR recognition of the M-MuLV gag peptide by CD8⁺ cells since (i) the CDR1β region has been shown to contact the C-terminal residues of the antigenic peptide in crystallographic studies of TCR-MHC class I-peptide complexes (17–19), (ii) CDR1β polymorphism in the Vβ10 gene segment has a dramatic influence on TCR recognition of the immunodominant H-2K^d-restricted HLA-CW3 epitope by CD8⁺ T cells (4), and (iii) CDR1β polymorphism of Vβ3 has already been shown to dramatically affect TCR recognition of a dominant pigeon cytochrome c peptide by MHC class II-restricted CD4⁺ T cells (16). Thus, the failure of M-MuLV-immune CTL from Vβ^b mice to use Vβ3 or Vβ17 is due to structural differences in these gene segments between the Vβ^a and Vβ^b alleles rather than an intrinsic preference for utilization of Vβ5.2 within the Vβ^b haplotype.

In order to directly compare the levels of utilization of Vβ3, Vβ5.2, and Vβ17 in the CD8⁺-T-cell response to the M-MuLV gag epitope, we used (B6 × B6.Vβ^a)F₁ mice in which all three Vβ domains are expressed. Analysis of the TCR repertoire of individual F₁ mice revealed a clear hierarchy in Vβ utilization. Thus, Vβ17 was used most frequently by responding CD8⁺ CD62L⁻ cells whereas Vβ3 (and especially Vβ5.2) were used to much lesser extents. Several possible explanations for this hierarchal Vβ usage can be considered. First, it is possible that Vβ17-bearing TCR have a higher affinity for the M-MuLV gag peptide than Vβ3- or Vβ5.2-bearing TCR. Although difficult to test directly, this explanation is, however, not supported by the comparable peptide dose-response curves of gag-specific CTL clones expressing Vβ17, Vβ3, or Vβ5.2. Second, it is possible that CD8⁺ T cells expressing Vβ17⁺ gag-specific TCR arise more frequently than Vβ3⁺ or Vβ5.2⁺ TCR with the same specificity, perhaps due to differential positive selection in the thymus. In this respect it is interesting that Vβ17 and Vβ3 chains of gag-specific TCR were quite heterogeneous in Jβ usage and CDR3 sequence but that Vβ5.2 chains were highly conserved (see below). These sequence data raise the possibility that Vβ17⁺ and Vβ3⁺ TCR recognizing the gag peptide are more frequent than gag-specific Vβ5.2⁺ TCR in the naive CD8⁺-T-cell population. Clearly, more direct experiments using MHC class I gag peptide tetramers in association with limiting dilution experiments will be required to address this issue.

Sequence analysis of the TCRα and -β chains utilized by CTL clones specific for the M-MuLV gag epitope revealed several important differences in the Vβ^a and Vβ^b haplotypes. Strikingly, TCRα and -β chains were absolutely conserved in Vβ^b mice, since all CTL clones analyzed utilized Vα3.2-Jα13 and Vβ5.2-Jβ1.4 with completely conserved CDR3α and CDR3β regions. These conserved TCRα and -β chains were representative of the M-MuLV-specific Vβ^b CTL population as a whole, since identical Vα3.2-Jα13 and Vβ5.2-Jβ1.4 junctional sequences were obtained by PCR from two independent polyclonal MLTC populations. In contrast, the TCR repertoires of M-MuLV gag-specific Vβ^a CTL clones were considerably more diverse. In particular, the TCRα chain was again strikingly conserved, with all Vβ^a CTL clones utilizing Vα3.2-Jα6 and a highly conserved (although not identical) CDR3α region. However, the TCRβ chains of Vβ^a CTL clones were much more diverse, since the two Vβ domains used (Vβ3 and Vβ17) were associated with three distinct Jβ segments and diverse CDR3β sequences.

These TCR sequence data are of interest in the context of a model proposing that the diversity of the TCR repertoire in response to a given foreign epitope is dependent upon the extent of homology between that epitope and self-determinants (9). According to this model, a consequence of self-tolerance will be that foreign epitopes that are highly homologous to self-peptides will elicit a restricted TCR repertoire but that epitopes unrelated to self will elicit a diverse TCR repertoire. In contrast, the data presented here indicate that the M-MuLV gag epitope CCLCLTVFL, which is not highly homologous to any expressed protein sequence (including endogenous retroviral leader sequences) in current databases (data not shown), can elicit a diverse repertoire of TCRβ chains in Vβ^a mice and a highly restricted β-chain repertoire in Vβ^b mice. Since these congenic mouse strains differ only at the Vβ locus itself, it seems probable that the diversity of the β-chain repertoire in this instance is determined by the availability of Vβ segments rather than by the extent of tolerance imposed by self-homology of the peptide epitope.

Finally, the finding that several structurally distinct TCR are able to recognize the same M-MuLV-encoded gag peptide associated with H-2D^b is consistent with growing evidence that TCR specificity is intrinsically highly cross-reactive or degenerate (27). Indeed, a recent crystallographic study has demonstrated that the same peptide-MHC complex can be recognized by two distinct TCR in the same orientation despite the fact that almost all the individual peptide-MHC contact residues differ (15). Conversely, a single TCR can recognize a wide variety of distinct peptide-MHC complexes with apparent high affinity, as in the case of the widely studied 2C TCR (34, 35). Whether such TCR degeneracy reflects selection by a common self-peptide in the thymus during development (21) remains to be established.