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International Immunology, Vol. 12, No. 3, 263-270, March 2000
© 2000 Japanese Society for Immunology

TCR Vß repertoire restriction and lack of CDR3 conservation implicate TCR–superantigen interactions in promoting the clonal evolution of murine thymic lymphomas

Gregorio Gomez1,4, Kimberly Z. Clarkin2, Ellen Kraig2, Anthony J. Infante3 and Ellen R. Richie1

1 Department of Carcinogenesis, and
2 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78724, USA
3 Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78724, USA

Correspondence to: E. Richie


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Thymic lymphoma development is a multistage process in which genetic and epigenetic events cooperate in the emergence of a malignant clone. The notion that signaling via TCR–ligand interactions plays a role in promoting the expansion of developing neoplastic clones is a matter of debate. To investigate this issue, we determined the TCR Vß repertoire of thymic lymphomas induced in AKR/J mice by either endogenous retroviruses or the carcinogen, N-methyl-N-nitrosourea (MNU). Both spontaneous and MNU-induced lymphomas displayed restricted Vß repertoires. However, whereas Vß6, Vß8 and Vß9 were expressed by a greater than expected frequency of MNU-induced lymphomas, Vß8, Vß7, Vß13 and Vß14 were over-represented on spontaneous lymphomas. The dissimilar TCR Vß profiles indicate that different endogenous ligands promote neoplastic clonal expansion in untreated and MNU-treated mice. Although the nature of these ligands is not clear, the lack of conservation in TCR ß chain CDR3 regions among lymphomas that express the same Vß segment suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, are likely to be involved in the selection process. The biased representation of lymphomas expressing Vß6-, Vß7- and Vß9-containing TCRs that recognize endogenous SAG is consistent with this hypothesis. The finding that Bcl-2 is expressed at high levels in spontaneous and MNU-induced lymphomas suggests that preneoplastic thymocytes may be resistant to SAG-induced clonal deletion. A working model is presented in which preneoplastic clones expressing TCRs that recognize endogenous SAG are selectively expanded as a consequence of sustained TCR-mediated signaling.

Keywords: AKR, J, Bcl-2, carcinogenesis, N-methyl-N-nitrosourea, thymocyte


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Lymphomagenesis is a multistage process in which accumulated genetic lesions promote the proliferation and/or survival of target cells that eventually generate an autonomous malignant clone (1). Both oncogene activation and tumor suppressor gene inactivation are implicated in the development of murine thymic lymphomas induced by radiation, chemical carcinogens or endogenous retroviruses (2–6). Although the specific genetic aberrations vary depending on the etiologic agent, most thymic lymphomas develop after a relatively long latency and consist of mono- or oligoclonal expansions indicating that rare preneoplastic clones are selected to complete the transformation process (7–10). Epigenetic factors also may play a role in selecting preneoplastic cells for continued tumor progression. For example, it has been proposed that viral antigen-mediated ligation of TCR on target cells activates a mitogenic signaling pathway that promotes retroviral-induced lymphomagenesis (11–13). The relevance of TCR-mediated signaling is supported by a recent report demonstrating that expression of a TCR{alpha} transgene was associated with spontaneous thymic lymphoma development which was further enhanced in mice that expressed both {alpha} and ß TCR transgenes (14). Alternatively, non- mitogenic signals induced by TCR–ligand interactions may operate on preneoplastic clones in a process analogous to the positive and negative selection events that occur during normal T cell development (15,16). Consistent with this notion, negative selection was reported to influence thymic lymphoma development in H-Y TCR transgenic male mice (17).

We explored the issue of TCR-mediated clonal selection during lymphoma development by analyzing the TCR Vß repertoire and ß chain complementarity-determining region 3 (CDR3) sequences from thymic lymphomas induced either by a chemical carcinogen or by endogenous retroviruses in the AKR/J mouse strain. AKR/J mice are highly susceptible to development of both spontaneous and N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas (18–21). Although both tumor types have similar histopathological characteristics and differentiation antigen phenotypes (19,22), they nevertheless are generated by distinct mechanisms. Spontaneous lymphomas occur in older (>6 months) AKR/J mice as a consequence of somatic integrations of endogenous recombinant murine leukemia viruses (MuLV) that activate the expression of cellular proto-oncogenes (reviewed in 23). In contrast, lymphomas generated by injecting MNU into young AKR/J mice appear after a relatively short (~3 months) latent period and arise independently of endogenous MuLV somatic integrations (24). Therefore, spontaneous and MNU-induced AKR/J lymphomas provide convenient models to compare the TCR Vß profile of thymic lymphomas induced by different etiologic agents in the same genetic background.

Supporting the notion that TCR-mediated signals confer a selective advantage to the expansion of early neoplastic clones (8,9,25), we report here that spontaneous and MNU-induced AKR/J thymic lymphomas present a restricted Vß repertoire. However, the distinct TCR Vß profiles indicate that unique intrathymic ligands are involved in retroviral- versus carcinogen-induced lymphomagenesis. Although the ligands responsible for skewing the Vß repertoire are unknown, the lack of conservation in CDR3 sequences cloned from lymphomas expressing identical TCR Vß gene products suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, promote the selection process. In this regard, it is interesting that a high frequency of MNU-induced lymphomas express the TCR Vß6 gene product since Vß6-expressing thymocytes are normally deleted in AKR/J mice as a result of negative selection by endogenous Mtv-7-encoded viral SAG (26). Taken together, the data support a model in which impairment of negative selection in rare neoplastic clones during lymphoma development promotes activation and selection of early neoplastic cells via TCRs that recognize endogenous SAG. This model is consistent with the observation that the anti-apoptotic protein Bcl-2 is highly expressed in MNU-induced and spontaneous lymphomas.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice and lymphoma induction
Male and female AKR/J mice were purchased from Jackson Laboratory (Bar Harbor, ME), and maintained in accordance with IACUC and institutional guidelines in the animal facility at the M. D. Anderson Science Park Research Division. Mice (4–6 weeks old) were injected with a single i.p. dose of 75 mg/kg MNU or maintained untreated until they developed spontaneous lymphomas at >6 months of age. Mice were sacrificed when lymphomas were evident or at the indicated time post-MNU treatment.

Immunofluorescence and flow cytometry
The following mAb were obtained from PharMingen (San Diego, CA): phycoerythrin (PE)–-anti-CD4 (RM4-5), FITC– or CyChrome–anti-CD8 (53–6.7), and biotinylated anti-CD69 (H1.2F3), anti-CD3 (145-2C11), anti-CD24 (M1/69), anti-CD25 (7D4), CD16/32 (Fc{gamma}RIII/II, 2.4G2) anti-Vß6 (RR4-7), antiVß8.1/8.2 (MR5-2), anti-Vß14 (14–2) and appropriate isotype controls. In addition, the following hybridoma supernatants were used in indirect immunofluorescence (IF): Vß3 (KJ25), Vß5 (MR9-4), Vß6 (RR47.15), Vß7 (TR3-10), Vß8 (F23.1), Vß9 (MR10-2), Vß11 (RR3-15), Vß13 (MR12-4) and Vß14 (14.2), which were detected by FITC-conjugated second-step reagents purchased from Jackson ImmunoResearch (West Grove, PA). For three-color IF analysis cells in HBSS containing 1% BSA and 0.1% sodium azide were incubated with directly conjugated antibodies on ice for 30 min followed by three washes and fixed in 1% paraformaldehyde. Binding of biotinylated antibody was detected by allophycocyanin- conjugated strepavidin (APC–SA) (Molecular Probes, Eugene, OR). For intracytoplasmic staining Bcl-2 cells were washed with PBS containing 0.03% Saponin followed by cell membrane permeabilization with 0.3% Saponin in PBS for 15 min at 25°C. The cells were washed twice with 0.03% Saponin and stained with 1 µg of anti-Bcl-2 (3F11) or an isotype control (PharMingen) followed by staining with FITC-conjugated goat anti-hamster IgG. Stained cells were analyzed with a Coulter Epics Elite flow cytometer (Miami, FL) equipped with an argon laser (488 nm) for FITC and PE excitation, and a helium neon laser (633 nm) for APC–SA excitation. Data were collected on 10–20x103 viable cells using a four-decade log amplifier and stored in list mode for subsequent analysis using Coulter (Miami, FL) Elite Software.

Southern blot analysis
DNA (10 µg) from individual MNU-induced and spontaneous thymic lymphomas was digested with HindIII and analyzed on Southern blots were hybridized with a randomly primed probe (27) that recognizes the entire Jß2 gene segment as previously described (7).

RT-PCR
Total RNA was isolated from individual lymphomas using the TRIzol Reagent (Gibco/BRL, Grand Island, NY), and cDNA was synthesized with MMLV reverse transcriptase (Gibco/BRL) using oligo(dT)12–18 primers (Gibco/BRL). Each PCR reaction was carried out in 100 mM Tris–HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 and 0.1% gelatin using 150 ng of one Vß-specific primer (Vß1–20) and one common Cß primer. The denaturation, annealing and extension cycles were: 1x(92°C, 5min; 37°C, 1min; 70°C, 2min), 3x(92°C, 1min; 37°C, 1min; 70°C, 2min), 2x(92°C, 1min; 42°C, 1min; 70°C, 2 min), 30x(92°C, 1 min; 55°C, 1 min; 70°C, 2 min) and 1x(92°C, 1 min; 55°C, 1 min; 70°C, 7 min). The primer sequences were identical to those described by Kraig et al (28). They were designed with 5' restriction sites to facilitate subsequent cloning steps.

CDR3 cloning and sequencing
After digestion with HindIII and EcoRI enzymes, the PCR products were cloned into pUC19 vector as described elsewhere (29). Briefly, the digested products were electrophoresed on a 2% agarose gel containing ethidium bromide into a trough filled with 15% polyethylene glycol (28). The DNA was collected while visualizing with a long-wave UV lamp. The purified PCR products were ligated into 100 ng of EcoRI- and HindIII-digested pUC19 vector using T4 DNA ligase (New England Biolabs, Beverly, MA) at 16°C. Plasmids were isolated from transformed DH-5{alpha} cells using the Wizard Plus DNA purification system from Promega (Madison, WI). DNA sequencing was carried out with the Sequenase version 2.0 DNA sequencing kit (United States Biochemical) using the forward M13 and reverse M13 primers. Only sequences with matching forward and reverse sequence are reported.

Statistical analysis
Statistical analysis of TCR Vß gene product representation on normal thymocytes compared to thymic lymphoma cells was performed using the exact binomial test (30).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The Vß repertoire is differentially biased in MNU-induced and spontaneous lymphomas
Primary MNU-induced and spontaneous AKR/J thymic lymphomas express similar differentiation antigen phenotypes. The majority of both types of lymphomas were double positive or expressed high levels of CD8 with variable levels of CD4. CD3 expression was generally at high to intermediate levels (Fig. 1Go). All lymphomas analyzed contained TCR ß gene rearrangements (Fig. 2Go) and expressed TCR {alpha} chain mRNA (data not shown). The discrete pattern of Jß2 rearrangements detected by Southern blot analysis indicates that most MNU-induced and spontaneous lymphomas are monoclonal. However, as illustrated in lane 4 of Fig. 2Go, oligoclonal lymphomas were occasionally observed.



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  		Fig. 1. Differentiation antigen phenotype of representative MNU-induced and spontaneous thymic lymphomas. Expression of CD4, CD8 and CD3 on thymocytes from untreated 3-month- old AKR/J mice is compared with two representative MNU-induced lymphomas and one spontaneous lymphoma. Dot-plots were obtained by staining cells with PE-conjugated anti-CD4 and FITC-conjugated anti-CD8. The histograms show the immunofluorescence profiles obtained by staining cells with biotinylated anti-CD3 followed by APC–SA (open histograms) or the second step alone (shaded histograms).

 


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  		Fig. 2. Comparison of Jß2 gene rearrangements in MNU-induced and spontaneous thymic lymphomas. DNA from normal thymus (NT), MNU-induced lymphomas (lanes 1–4) and spontaneous (lanes 5–8) lymphomas was digested with HindIII, and Southern blots were hybridized with a probe specific for the Jß2 gene cluster (27). The arrow indicates the position of the unrearranged 4.4 kb germline band.

 
Given that these lymphomas express readily detectable levels of CD3 and are mono- or oligoclonal, we asked if there was a random or biased distribution of TCR Vß specificities expressed by either type of lymphoma. The TCR repertoires expressed by primary MNU-induced or spontaneous lymphomas were determined by IF analysis of reactivity with a panel of mAb that recognize distinct TCR Vß chains. Thirty-two of 61 (52%) MNU-induced lymphomas and 16 of 24 (67%) spontaneous lymphomas expressed a Vß determinant detected by the mAb panel. Figure 3Go presents the expected versus observed frequency of lymphomas expressing the indicated Vß specificities. Expected values are based on the percentage of thymocytes in normal young AKR/J mice that express the indicated Vß determinants. If all thymocytes, regardless of Vß specificity, were equally likely to undergo neoplastic conversion, then the percentage of lymphomas expressing a given Vß determinant would reflect the frequency of normal thymocytes expressing that Vß determinant.



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  		Fig. 3. The expected versus observed frequency of MNU-induced and spontaneous thymic lymphomas expressing specific Vß determinants. Single-cell suspensions from individual MNU-induced (A) or spontaneous (B) AKR/J lymphomas were stained with a panel of anti-Vß specific mAb and analyzed by indirect immunofluorescence. The black bars represent the expected frequency calculated as the mean ± SD of the percentage of thymocytes from normal AKR/J mice (n = 4) that bind a particular anti-Vß mAb. The hatched bars represent the observed frequency of lymphomas that bind the indicated anti-Vß mAb. Asterisks mark the Vß determinants that were represented on lymphomas at significantly greater than expected frequencies.

 
The data in Fig. 3Go show that MNU-induced and spontaneous lymphomas express a biased TCR Vß repertoire. Three Vß gene segments were expressed at a greater than expected frequency in the MNU-induced lymphomas: Vß6 (P < 0.0001), Vß8 (P < 0.04) and Vß9 (P < 0.001). Strikingly, 16% of MNU-induced lymphomas expressed TCRs utilizing the Vß6 segment, whereas Vß6 was expressed on only ~3% of the normal thymocyte population. Interestingly, although the Vß repertoire was also restricted in spontaneous lymphomas, the pattern of Vß usage was distinct from that observed in MNU-induced lymphomas. Approximately 30% of spontaneous lymphomas expressed Vß8 (P < 0.006), but there was not an over-representation of tumors that expressed Vß6. There was also a greater than expected frequency of spontaneous lymphomas that expressed Vß7 (P < 0.0004), Vß13 (P < 0.01) and Vß14 (P < 0.004). Taken together, these data demonstrate that the Vß repertoire is differentially biased in MNU-induced and spontaneous lymphomas arising in the same genetic background.

The IF analysis demonstrated that only a single Vß determinant was expressed by the vast majority of MNU-induced and spontaneous lymphomas. However, the fraction of Vß+ lymphoma cells within a given tumor was highly variable. RT-PCR analysis verified that a single Vß specificity was expressed regardless of the percentage of Vß+ cells. For example, Fig. 4Go shows that only the Vß6 primer generated a PCR product from cDNA of lymphoma 8560 which contained 97% Vß6+ cells by IF analysis. Similarly, Vß6 was the predominant PCR product amplified from lymphoma 8596 which contained a minority (23%) of Vß6+ cells by IF analysis. Lymphoma 8576 was one of the rare apparently biclonal tumors that reacted with two anti-Vß reagents such that 25% of the cells stained positively for Vß6 and 28% stained positively for Vß8. Consistent with the IF data, Vß6 and Vß8 PCR products were amplified from lymphoma 8576.



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  		Fig. 4. TCR Vß gene products expressed in MNU-induced lymphomas. TCR Vß gene segments were amplified from normal AKR/J thymocytes or MNU-induced lymphomas 8560, 8596 or 8576 by RT-PCR using primers specific for 20 different Vß gene segments and one common Cß primer as described in Methods.

 
Negative selection is generally operative in MNU-treated mice
In the AKR/J strain, the endogenous Mtv-7 SAG induces deletion during negative selection of thymocytes expressing TCR containing Vß6, Vß7, Vß8.1 or Vß9 gene products (26,31,32). Intriguingly, these Vß specificities correspond to several that are over-represented in MNU-induced thymic lymphomas suggesting that expression of Mtv7-reactive TCR may confer a selective advantage to developing neoplastic clones as opposed to inducing their clonal deletion. To determine if MNU generally impairs negative selection, thereby allowing autoreactive clones to escape deletion, we determined the percentage of cells that express Vß6 in CD4+ single-positive (SP) thymocytes and lymph node T cells obtained 5 weeks after MNU injection. At this early stage of the latency period, thymocyte cellularity and subset distribution are similar to age-matched untreated littermates, and overt lymphoma is not histologically detectable (33). For comparison, the percentage of cells that expresses Vß14 was evaluated since Mtv7 does not react with Vß14-containing TCRs. The data in Table 1Go show that there is a paucity of Vß6+ thymocytes and lymph node cells in the CD4 SP compartment of both untreated and MNU-injected mice. Similarly, the percentage of Vß14+ thymocytes or peripheral T cells was not altered by MNU treatment. These results demonstrate that negative selection of Vß6-expressing T cells is not generally compromised in AKR/J mice as a result of MNU treatment.


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  		Table 1. Frequency of CD4+8- thymocytes and lymph node cells that express Vß6 or Vß14
 
Bcl-2 is highly expressed in MNU-induced and spontaneous lymphomas
Considering that negative selection is operative in most thymocytes during the latent period, it is possible that MNU-induced genetic alterations are sustained by a rare preneoplastic clone(s), and thereby provide a survival advantage that promotes clonal expansion and lymphomagenesis. Since Bcl-2 not only enhances thymocyte survival, but also has been shown to collaborate with activated oncogenes in promoting lymphomagenesis, we compared the relative expression levels of this anti-apoptotic protein in normal thymocytes, MNU-induced and spontaneous lymphomas. As shown in Fig. 5Go, Bcl-2 is expressed at relatively high levels in both types of lymphomas compared to normal thymocytes. Similar results were obtained with three additional MNU-induced and three additional spontaneous lymphomas.



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  		Fig. 5. Comparison of Bcl-2 expression in normal thymocytes, MNU-induced and spontaneous thymic lymphomas. Intracellular Bcl-2 expression was detected using an indirect IF assay in normal thymocytes (dashed line), a representative MNU-induced lymphoma (thick solid line) and a representative spontaneous lymphoma (thin solid line). Permeabilized cell suspensions were incubated with anti-Bcl-2 mAb or isotype-matched control mAb followed by FITC-conjugated anti-Ig. The dotted line shows the isotype control profile obtained for normal thymocytes. An identical pattern was observed for the lymphoma isotype controls.

 
CDR3 sequences are not conserved among Vß6- or Vß8-expressing thymic lymphomas
The biased Vß repertoire observed in MNU-induced and spontaneous lymphomas suggests that signals mediated by TCR interactions with endogenous peptide or SAG confer a selective advantage for continued evolution of neoplastic thymocytes. It is generally agreed that peptides presented in the peptide binding groove of MHC molecules are recognized by the variable regions of both TCR {alpha} and ß chains. Peptide binding specificity is primarily a function of the CDR3 regions which comprise the junctional domains encoded by V, D (in ß chain) and J nucleotides (39). In contrast, SAG bind to the lateral surface of MHC class II molecules and interact with the HV4 region of the TCR ß chain, although recent studies have shown that additional sites can contribute to SAG binding (40,42). If TCR interactions with conventional peptides were involved in promoting the clonal expansion of neoplastic thymocytes during lymphomagenesis, then conservation of the ß chain CDR3 amino acid sequence would be expected among different lymphomas that express the same TCR Vß gene segment. On the other hand, conservation among CDR3 segments would not be expected if SAG were involved in the selection process. To distinguish between these alternatives, we determined the CDR3 nucleotide sequence of the TCR ß chain of Vß6+ or Vß8+ MNU-induced lymphomas and Vß8+ spontaneous lymphomas. Table 2Go shows that there is no apparent conservation in the length or deduced amino acid sequence of the CDR3 segments expressed in either Vß6+ or Vß8+ lymphomas. Moreover, there is no conservation in Jß gene segment usage among the lymphomas analyzed. The lack of conservation observed within the CDR3 region is most compatible with the hypothesis that the high frequency of Vß6+ and Vß8+ lymphomas is due to a selection process mediated by endogenous SAG as opposed to conventional peptides. However, the finding that Vß8.2 is the most frequently utilized Vß8 family member in MNU-induced and spontaneous lymphomas suggests that another SAG instead of, or in addition to, endogenous Mtv7 may be involved.


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  		Table 2. TCR ß-chain junctional regions from MNU-induced and spontaneous lymphomas
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The significance of antigen receptor-mediated signaling events in lymphomagenesis is an intriguing, but controversial, issue. A long-standing hypothesis proposed by Weissman and colleagues submits that persistent signaling of target cells via TCR interactions with viral antigens plays a role in retroviral-induced thymic lymphoma development (11–13). Although V(D)J recombination and TCR expression are dispensable for the generation of thymic lymphomas in irradiated and p53-deficient RAG-1–/– and SCID mice (43–45), TCR expression has been shown to promote lymphoma progression in certain TCR transgenic models (14,44). Furthermore, earlier investigations suggested that negative or positive selection plays a role in lymphomagenesis (14,17). In fact, positive selection was presumed to play a role in the preferential Vß utilization observed in thymic lymphomas induced by radiation, retroviruses or expression of a c-myc transgene (8,9,46,47). These reports suggest that TCR expression, while not required for lymphoma development, promotes lymphoma progression.

The present investigation supports the notion that TCR engagement is one epigenetic factor that contributes to the evolution and selection of neoplastic T cell clones. The biased TCR Vß repertoire expressed by spontaneous and MNU-induced lymphomas suggests that selection of preneoplastic thymocytes for continued expansion is not a random process, but rather reflects the preferential progression of clones that express particular TCR Vß determinants. A previous analysis of the clonal evolution of MNU-induced lymphomas revealed that trisomy of chromosome 15 as well as K-ras mutations were detected early in the latent period, prior to the emergence of a dominant clone (48). These observations are consistent with the notion that signaling via TCR expressed on aberrant preneoplastic cells is a secondary epigenetic event that promotes the selection and emergence of a dominant clone.

Based on the lack of conservation in CDR3 length and amino acid sequence among different lymphomas expressing the same TCR Vß segment, we propose that endogenous SAG, rather than endogenous peptide, contributes to the selective expansion of neoplastic clones expressing Vß6 or Vß8.2. Of course, it is possible that analysis of CDR3 regions from lymphomas expressing different TCR Vß segments would support a role for endogenous peptides in lymphoma development. Although the nature of the endogenous ligands that promote neoplastic expansion is not known, the over-representation of Vß8.2 in both spontaneous and MNU-induced lymphomas suggests that a common ligand in the thymic microenvironment selects Vß8.2+ cells for sustained expansion regardless of etiology. Also, the fact that Vß8.2 rather than Vß8.1 is over-represented in AKR/J lymphomas indicates that Mtv7 is not responsible for biased Vß8.2 usage. On the other hand, Mtv7-encoded SAG may be involved in promoting the high frequency of Vß6-expressing MNU-induced lymphomas. If so, it is unclear why Vß6 over-representation was not observed in spontaneous lymphomas. Possibly, treatment with the carcinogen increased expression of Mtv7-encoded SAG or induced the expression of a novel Vß6 binding SAG.

MNU treatment did not result in the escape of mature SP Vß6-expressing T cells into the periphery, demonstrating that negative selection remains operative during the latent period prior to lymphoma development. Nevertheless, the over-representation of lymphomas expressing `forbidden' Vß segments suggests that oncogenic alterations which impede deletion induced by endogenous SAG are an important component of the multistage pathway leading to lymphoma development. It is likely that rare preneoplastic clones sustain MNU-induced genetic lesions that block apoptosis after TCR-mediated interactions with Mtv or other endogenous ligands. Since Bcl-2 is poorly expressed in normal DP thymocytes, the high levels of Bcl-2 observed in the lymphomas suggest that aberrant regulation of this protein may confer resistance to SAG-induced deletion (49,50). Although there are reports that Bcl-2 protects autoreactive thymocytes from deletion by superantigens or endogenous peptides (51–53), other studies (54,55) failed to demonstrate that Bcl-2 impairs the negative selection process. Recently, it was reported that overexpression of a bcl-2 transgene promoted the production of SP mature T cells in mice that expressed a class I-restricted transgenic TCR on a RAG-1–/– background (56). Furthermore, enforced bcl-2 expression inhibited peptide-induced negative selection in fetal thymic organ culture. Therefore, it is possible that deregulated bcl-2 expression subverts an apoptotic pathway. Taken together, the data suggest a working model in which viral or carcinogen-induced genetic alterations impair negative selection in rare thymocyte targets. Furthermore, a subset of preneoplastic target cells expressing TCRs that recognize endogenous SAG is preferentially selected for expansion as a consequence of sustained signaling via TCR–SAG interactions.


   Acknowledgments
 
The authors gratefully acknowledge the statistical expertise of Dr Dennis Johnston, the skilled technical assistance of Marina Holloway and the secretarial assistance of Carrie McKinley. This work was supported by National Institute of Health Grants CA37912 (to E. R. R.), a grant from the Bruce McMillan Jr Foundation and NIEHS Center grant ES07784.


   Abbreviations
 
APC allophycocyanin
CDR3 complementarity determining region 3
IF immunofluorescence
MNU N-methyl-N-nitrosourea
MuLV murine leukemia viruses
PE phycoerythrin
SA streptavidin
SAG superantigen
SP single positive

   Notes
 
4 Present address: Building 10, Room 11N256, LI/NIAID/NIH, 10 Center Drive, Bethesda, MD 20892, USA Back

Transmitting editor: J. P. Allison

Received 25 August 1999, accepted 3 November 1999.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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