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The Central Tolerance Response to Male Antigen in Normal Mice Is Deletion and Not Receptor Editing ¹

Department of Laboratory Medicine and Pathology and Center for Immunology, University of Minnesota, Minneapolis, MN 55455

	Abstract

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It is widely accepted that developing T cells can undergo clonal deletion in the thymus in response to a high affinity self-Ag. This is largely based on studies of TCR transgenics. However, encounter with high affinity self-Ag can also result in receptor editing in TCR transgenic models. Because all TCR transgenics display ectopic receptor expression, the tolerance mechanism that predominates in normal mice remains an open question. When self-Ag drives receptor editing during T cell development, one expects to find in-frame, self-reactive TCR joins on TCR excision circles (TRECs), which are the products of secondary V/J recombination in the TCR locus. Such joins are not expected if clonal deletion occurs, because the progenitor cell would be eliminated by apoptosis. To test the relative utilization of receptor editing vs clonal deletion, we determined the frequency of in-frame, male-specific joins on TRECs in male and female HY transgenic mice. In comparison with female HY transgenic mice, our analysis showed a lower frequency of TRECs with male-reactive V17J57 joins in male mice. Thus, it would appear that receptor editing is not a predominant tolerance mechanism for this self-Ag.

	Introduction

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During T lymphocyte development several molecular processes act to generate TCR diversity, including somatic rearrangement of the TCR genes, the addition of template-dependent or -independent nucleotides, and the combinatorial association of heterodimeric TCR - and -chains (1). Once a heterodimeric receptor is formed, cellular selection and survival signals play a critical role in the further development (2). A progenitor is included (positive selection) when there is a low affinity interaction of the TCR with a self-peptide/MHC ligand or is excluded (negative selection) when it is a high affinity interaction. Thus, an effective T cell repertoire is shaped by the interplay of molecular and cellular processes.

With identification of the lymphoid-specific recombination activating gene-1 (RAG1) ³ and RAG2 and analysis of TCR- and TCR-deficient mice, the complex nature of the interaction and timing of expression of proteins involved in generating receptor diversity became evident. RAG expression is turned on in immature CD25⁺CD4^-CD8^- (double negative (DN)) thymocytes (3, 4). At this stage, the TCR locus undergoes recombination, being accessible as a result of lineage- and stage-specific factors (1). Productive rearrangement and expression of a TCR-chain and its association with the invariant -chain (pre-T (pT)) are the first checkpoints in establishing TCR clonality. Following selection, RAG activity is down-regulated, and the TCR locus ceases to undergo further rearrangement (5), imposing fairly efficient TCR allelic exclusion. As the thymocyte traverses into the CD4⁺CD8⁺ (double positive (DP)) stage, RAG is re-expressed, and the TCR locus now becomes accessible to rearrangement (6). However, unlike the strict allelic exclusion observed for the locus, the locus is subject to extensive successive (secondary) rearrangement, resulting in either a changed TCR specificity or the expression of two different TCR (7, 8). In fact, the TCR locus appears to be specifically suited for sequential V gene rearrangements, as it is composed of 98 upstream V genes and 60 downstream J genes spanning 1.5 Mb (9). Studies point to a bidirectional and coordinated mechanism of sequential rearrangement where 3'V genes are initially rearranged to 5'J genes, and subsequently the more 5'V genes are rearranged to more 3'J genes (8, 10, 11, 12). Almost all T cells have both TCR alleles rearranged. In fact, 30% of peripheral T cells have productive rearrangements on both TCR alleles (11), leading to a high percentage of T cells expressing two TCR-chains, a phenomenon referred to as allelic inclusion (13, 14). The potential of the TCR-chain locus for successive gene rearrangement suggests an inherent mechanism by which a T cell could escape death by neglect or clonal deletion (15).

That T cells use secondary rearrangement to escape death by neglect was clearly established by the finding of increased endogenous TCR gene rearrangement in TCR transgenic (Tg) mice on a nonselecting background (16). However, T cells can apparently escape death by clonal deletion using secondary rearrangement as well. This phenomenon has been referred to as receptor editing. Wang et al. (17) reported that T cells from a mouse with the cytochrome-specific TCR-chain knocked into the J locus (2B4KI) had changed receptor specificity by deleting the 2B4KI in the presence of Ag. We also described receptor editing in T cells as an Ag-driven process, using the OT-I Tg mouse, which expresses a TCR specific for an OVA peptide, presented in the context of the class I MHC (K^b) molecule. Ex vivo experiments using fetal thymic organ cultures and exogenous OVA peptide clearly demonstrated deletion of DP OT-I thymocytes consistent with clonal deletion (18). Surprisingly, in vivo expression of the OVA peptide in thymic epithelial cells did not result in efficient deletion, but yielded a large population of T cells expressing endogenous V-chains (19). The importance of recombination in promoting development was clear, as mature thymocytes were absent in double-Tg mice on a RAG1-deficient background.

In contrast, a recent report by Buch et al. (20) described the lack of receptor editing in the HY model. The approach taken by this group was similar to that of Wang et al. (17), in which an Ag-specific -chain was knocked into the locus and thus was subject to the gene rearrangement process. In this sophisticated knockin mouse, the locus was manipulated such that the TCR region was replaced with a male reactive V/J join in the 5' region of the J locus, thus mimicking the end product of natural V/J recombination appropriately. The targeted locus (I) in combination with the appropriate TCR Tg created a mouse with monoclonal potential for male reactivity. In theory, male reactivity could be eliminated by secondary recombination in this model, because recombination would excise the male reactive V/J segment. Thus, these mice represent a good model for studying Ag-driven receptor editing. Nonetheless, a striking increase in the occurrence of secondary rearrangement in the presence of male Ag was not observed, suggesting that receptor editing does not occur (20). However, a significant concern regarding the interpretation of this study is the ectopic or early expression of the TCR-chain. Normal mice first express a TCR-chain at the DP stage, but TCR Tg express it at the earlier DN stage. Early expression is observed to varying degrees in all TCR Tg strains and contributes to marked abnormalities, such as reduced cellularity due to competition with the pre-TCR (21, 22, 23), architectural disorganization (24), and a high level of mature DN cells (25). High surface expression of the TCR-chain was observed in CD25⁺ (stage III) DN progenitors in HYI/ mice, as it was in conventional HY mice (20) (data not shown). In the case of both conventional and HYI/ mice, there is a strong possibility that male Ag induces deletion of progenitors at the DN stage (26). In this case, one would not expect efficient receptor editing, because the TCR locus is not available for recombination at the DN stage, as outlined above. In contrast, the TCR in OT-I mice is rather poorly expressed on DN progenitor cells (21) (data not shown). Because relatively efficient receptor editing was observed in OT-I mice, we hypothesized that ectopic or early expression of TCR transgenes might be masking a natural receptor editing response to self-Ag in the I/ and other TCR Tg models. Our approach was therefore to determine whether self-Ag-driven receptor editing could be observed in HY mice, where TCR-chains are not expressed until the DP stage.

	Materials and Methods

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Animals

HY/E^+/ mice were generated by crossing HY Tg mice (27) (provided by H. von Boehmer) with E^/ mice (28) (provided by B. Sleckman), maintained under specific pathogen-free conditions, and sacrificed following protocols approved by the Institutional Animal Care and Use Committee at University of Minnesota. Mice were typed by PCR using the following primers: HYs-1, 5'-cacatggaggctgcagtcac-3'; and HYs-2, 5'-gtttctgcactgttatcacc-3' for HY; and 5'-gctttgggtaaaagccacatgggta-3' and 5'-agtacctgttatgggcgaccccttt-3' for E.

T cell purification

Lymph nodes (LN) were harvested and pooled from HY/E^+/ mice and incubated with FITC-labeled anti-V2 (B20.1) Ab in MACS buffer (PBS containing 0.5% FCS and 2 mM EDTA) for 5 min. Cells were washed with MACS buffer and incubated with beads coupled to an anti-FITC Ab according to the manufacturer’s suggestion (Miltenyi Biotec, Auburn, CA). T cells were enriched for the V2⁺ population by purifying them twice over tandem LS⁺ columns at 4°C. DNA from V2⁺-enriched cells (0.8–1 x 10⁶) was extracted using the Easy DNA kit (Invitrogen, San Diego, CA) following the manufacturer’s protocol.

Flow cytometric analysis

Unpurified and V2⁺-enriched T cells were incubated for 1 h on ice in staining buffer (PBS, 1% FCS, and 3 mM sodium azide) with fluorochrome-conjugated Abs to V2, V8, Thy1.2, CD4, or CD8 (BD PharMingen (San Diego, CA) or e-Biosciences (San Diego, CA)). Data were acquired using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and were analyzed using FlowJo software (TreeStar, San Carlos, CA).

Polymerase chain reaction

DNA (100–200 ng) containing T cell receptor excision circles (TRECs) from V2⁺ cells was amplified by PCR using the primers AV17EcoRI (5'-gggaattcacaccgttgttaaaggcacc-3') and AJ57BamHI (5'-ggggatcctgtcccctccccaaagatga-3'). Restriction sites used for cloning are underlined. PCR parameters were 94°C (180 s), followed by 94°C (45 s), 56°C (45 s), and 72°C (105 s), for 36 cycles. A minimum of three separate PCR reactions were run for each template, and the product (190 bp) was excised from an agarose gel and purified using the QiaQuick Gel Extraction kit (Qiagen, Valencia, CA). The purified product was digested with BamHI and EcoRI (Invitrogen) and cloned into pUC19. DH5 electrocompetent cells (Invitrogen) were transformed and plated on Luria-Bertoni solution-containing ampicillin plates. Individual colonies were picked, and plasmid DNA was purified using a Miniprep Spin Kit (Qiagen). Sequencing was performed with the AV17EcoRI primer at the Advanced Genetic Analysis Center (University of Minnesota).

	Results and Discussions

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The low precursor frequency of Ag-reactive T cells does not favor analysis of deletion and receptor editing in normal mice. Thus, for these experiments we chose to use a TCR Tg strain because it has an increased frequency of Ag-specific T cells. Although one might argue that these are not normal mice, they do not display the marked abnormalities of TCR Tg strains (21, 24). Indeed, endogenous TCR-chains are produced and expressed at the DNIII stage of thymic development; thus, the expression of a Tg -chain at this stage is not considered ectopic. Importantly, in such mice a heterodimeric TCR is not expressed until the DP stage, since it relies on the endogenous TCR locus recombination process. Thus, such mice allow determination of the significance of receptor editing in the context of appropriate receptor timing.

The HY male Ag-specific TCR -chain Tg mouse (27) provided an ideal model system for these studies. Bouneaud and colleagues (29) extensively analyzed the TCR-chain usage of male-reactive CD8 T cells in HY Tg mice using D^b/male Ag tetramers. Their results showed a highly focused repertoire composed of T cells expressing a TRAV17 TCR-chain (referred to previously as V9). Strikingly, sequence analysis of the complementary-determining region 3 (CDR3) junctions from tetramer-positive T cells containing a V17J57 rearrangement revealed the presence of CDR3 junctions encoding 10 or 11 aa in female mice, but not in males. It was therefore suggested that T cells expressing a high affinity, male-reactive V17J57 -chain with a CDR3 of 10 or 11 aa are deleted from the repertoire of male mice through negative selection. However, the absence of this population of T cells in male mice could also be a consequence of editing of the male-reactive TCR and subsequently selection of T cells expressing a different TCR. Thus, we believed it was important to distinguish between these two possibilities.

The presence of in-frame VJ rearrangements on excision circles in thymocytes is evidence of secondary rearrangement at the locus (30). We therefore reasoned that if receptor editing occurs in response to self-Ag exposure during development, one would observe an increased frequency of self-reactive in-frame VJ joins on TRECs in the presence of self-Ag compared with that in its absence. In the HY Tg mouse, clonal deletion would predict a low and perhaps undetectable frequency of T cells containing male-reactive V17J57 joins on TRECs. On the other hand, if receptor editing were a major process in shaping the selected T cell repertoire, male-reactive V17J57 joins would be found more frequently on TRECs in male mice. In addition, the V17J57 rearrangement is predicted to be a primary or early rearrangement as TRAV17 is one of the most 3'V segments in the locus, and TRAJ57 is one of the most 5'J segments in the locus (9). Thus, an allele with this rearrangement would be predicted to easily undergo secondary gene rearrangement, resulting in the generation of TRECs with these joins. Finally, as the male Ag is expressed widely in all tissues (31), it is likely to be encountered at the DP stage when the progenitor is capable of secondary TCR locus rearrangement. Overall, the HY Tg mouse is a good model for deciphering the relative contribution of negative selection and receptor editing in shaping the TCR repertoire.

Approach to detect male-reactive V17J57 joins

The HY -chain is encoded by one of the most 3'V genes (TRAV17) rearranged to one of the most 5'J genes (TRAJ57) in the locus (9) (see Fig. 1). Thus, it is typical of those joins that occur early in the life span of a progenitor (12). A rearranged V17J57 join is therefore also readily subject to excision by secondary rearrangement and can be detected as a TREC within the T cell (Fig. 1). TRECs are relatively stable in the cell, being diluted primarily by cell division, and can be easily detected in naive T cells because little cell division occurs after thymic selection (32). Thus, one can query the content of excision circles by using peripheral T cells. We sought to determine the frequency of in-frame, male-reactive joins on excision circles in peripheral T cells of male and female HY Tg mice. However, current protocols for the isolation of TRECs primarily enrich, rather than purify, and it is difficult to exclude contamination with genomic DNA. Thus, we adopted a strategy that involves rigorously purifying V2⁺ T cells (Fig. 1). Such T cells have undergone recombination using one of the 6 V2 subfamily members (TRAV14), all of which reside upstream of TRAV17 in the TCR locus (9). Any in-frame V17 rearrangements therefore must either have been on an excision circle or the alternate allele. Second, to distinguish these two possibilities, we used mice that were heterozygous for a mutation in the -chain enhancer (E) (28). This cis-acting mutation profoundly impairs rearrangement of the locus only on that chromosome. Thus, in V2⁺ cells from such mice, V17J57 joins largely reflect a primary rearrangement event, which was excised by secondary rearrangement between a 5'V2 and a 3'J (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Experimental strategy. HY Tg mice were crossed to the -chain enhancer knockout mouse (E/) to generate HY, E+/ mice used in this study. The mouse -chain alleles are designated as 14a (recombination defective) and 14b (wild type). A primary rearrangement event can occur on the normal allele between V17 and J57. In the event of a secondary rearrangement, one of six upstream V2 segments (recently renamed V14) can combine with a downstream J segment (Jx), and the V17J57 rearrangement is excised as a TREC. Thus, V17J57 joins that are detected in V2+ T cells represent a primary rearrangement that was excised by secondary recombination. C, constant -chain. Arrows represent primers used to amplify the junction of V17J57 joins.

+/

+/

View larger version (13K): [in this window] [in a new window]   FIGURE 2. V2+ T cells purified from LN of male and female HY Tg express the V8-chain from the Tg. A, T cells purified from LN using a FITC-coupled anti-V2 Ab and anti-FITC-coupled magnetic beads are >99% pure (). , LN cells before purification (range, 5–8% V2+; data not shown). B, Greater than 99% of purified V2+ T cells express the Tg V8 chain.

+/

HY males have reduced occurrence of male-reactive TRECs

The relative occurrence of joins encoding high affinity, male-reactive TRECs in V2⁺ T cells isolated from male and female mice was used as the criterion to determine whether receptor editing occurred in the HY Tg mice. An increased occurrence in males compared with females would support receptor editing as a tolerance mechanism. Alternatively, a similar or reduced occurrence in males compared with females would support a deletion mechanism. The number of TRECs isolated are reported as a ratio of male-reactive V17J57 TRECs to out-of-frame sequences, as the occurrence of out-of-frame sequences are independent of selection pressures and thus should be equivalent in males and females. Our data showed a significant reduction in the ratio of male-reactive joins in male (0.025) compared with female (0.071) mice (Table I). Furthermore, using the criteria of closely conserved amino acid substitutions in the CDR3 junction, we also noted a significant reduction in the occurrence of male-reactive joins in male (0.062) compared with female (0.130) mice. Finally, if male reactivity were defined solely by CDR3 length of 10 or 11 aa, we observed a >1.5-fold decrease in male-reactive joins in males compared with females (29 vs 50; data not shown).

These differences are highly likely to reflect selection processes, since the overall frequency of in-frame to out-of-frame was not significantly different between males and females. Furthermore, the occurrence of joins with a CDR3 length of 9 aa was not different between males and females (see Table I). Thus, by all criteria examined, a lower occurrence of TRECs containing a rearranged V17J57 was found in male compared with female HY Tg mice. There is a concern that thymocyte encounter with male Ag during receptor editing could drive proliferation of the cell and thereby dilute TRECs. However, we think this is unlikely because proliferation does not occur in DP thymocytes undergoing positive or negative selection. Altogether these results support the interpretation that clonal deletion shapes the selected T cell repertoire specific to the male Ag.

The extent of reduction of male-reactive joins in male mice compared with female mice (2.2-fold) was not as striking as one might expect for robust clonal deletion. Six in-frame, male-reactive joins were detected on excision circles in male mice. Does this indicate that receptor editing happens on occasion? We think that it is not possible to make this conclusion from our data. An in-frame, male-reactive V17-containing receptor may have been replaced by secondary rearrangement before the receptor was assembled and expressed on the cell surface or before the progenitor encountered Ag. This, of course, could also occur in female mice and thus is inherently controlled for in our experimental design. In addition, positive selection of T cells expressing a selectable male-reactive TCR in female mice would reduce the number of T cells with male-reactive TRECs. Thus, it would be predicted that an even greater difference in the frequency of male-reactive TRECs would be observed between male and female mice on a nonselecting background.

The fact that we did not find an increased occurrence in male mice suggests that receptor editing is not a predominant central tolerance mechanism for this particular Ag. This finding therefore is consistent with studies that used monoclonal TCR Tg mice. However, it should be noted that our study did not identify the stage at which central tolerance occurs. It was concluded from studies of monoclonal mice that tolerance to male Ag occurs before or coincident with the expression of CD4 and CD8 during development (20). As we outlined in the introduction, standard monoclonal models have unusually early expression of the transgene and also suffer from nonphysiologic processes as a result of the grossly exaggerated frequency of responders. For these reasons it would be premature to conclude that normal self-reactive progenitors are deleted at the early DNDP stage. In fact, recent studies in which a peripheral T cell response to Ag was prevented (33, 34) or modified (35) found a reduced efficiency of DP deletion, suggesting that clonal deletion may normally occur at the late DPSP stage.

It also remains to be determined why Ag-specific receptor editing was so pronounced in some monoclonal models (17, 19), but not others (20). In our study of receptor editing in OT-I mice, we noted a correlation of receptor editing with Ag-induced TCR internalization in vivo (19). This led us to propose a model of receptor editing based on the absence of a basal signal to repress RAG (15, 19). Thus, one potential factor that could dictate whether receptor editing or clonal deletion occurs is the efficiency of Ag-induced internalization. Little is known about the biochemical basis of ligand-induced internalization, and it is not known whether different Ag receptors can exhibit different efficiencies. Thus, while this study shows that receptor editing does not substantially contribute to central tolerance for one male Ag, it may be involved in tolerance to other Ags.

	Acknowledgments

 
We thank Steve Jameson and other lab members for helpful discussion, P. Bousso for unpublished sequences of male-reactive joins, and H. von Boehmer and B. Sleckman for providing the HY and E^/ mice, respectively.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI50105 and AI35296, and Lupus Foundation of Minnesota Grant 10006802 (to P.O.H.).

² Address correspondence and reprint requests to Dr. Kristin Hogquist, Department of Laboratory Medicine and Pathology and Center for Immunology, University of Minnesota, Minneapolis, MN 55455. E-mail address: hogqu001{at}umn.edu

³ Abbreviations used in this paper: RAG1, recombination activating gene 1; CDR3, complementary-determining region 3; DN, double negative; DP, double positive; E, -chain enhancer; LN, lymph node; pT, pre-T; SP, single positive; Tg, transgenic; TREC, T cell receptor excision circle.

Received for publication June 20, 2003. Accepted for publication August 14, 2003.

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Top Abstract Introduction Materials and Methods Results and Discussions References

 

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