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Original Article |
yannoun{at}mail.rockefeller.edu
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/β genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRβ recombination in CD4–CD8–CD25+ thymocytes. In these cells, TCRβ expression leads to clonal expansion, RAG downregulation, and TCRβ allelic exclusion. At the subsequent CD4+CD8+ stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCR locus. This second wave of RAG expression is terminated upon expression of a positively selected /β TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4+CD8+ T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4+CD8+ cells but a 50–70% reduction in the number of mature CD4+CD8– and CD4–CD8+ thymocytes. TCR rearrangement is restricted to the 5' end of the J cluster and there is little apparent secondary TCR recombination. Comparison of the TCR genes expressed in wild-type or mutant mice shows that 65% of all /β T cells carry receptors that are normally assembled by secondary TCR rearrangement. We conclude that RAG reinduction in CD4+CD8+ thymocytes is not required for initial TCR recombination but is essential for secondary TCR recombination and that the majority of TCR chains expressed in mature T cells are products of secondary recombination.
Key Words: T cell receptor chain • gene rearrangement • regulation of gene expression • T cell receptor editing • recombination activating gene
In thymocytes, V(D)J recombination is initiated at the TCRβ locus in CD4–CD8– double negative (DN) T cells 16. Once a TCRβ chain is expressed it combines with pre-T
In DP thymocytes V(D)J recombination is targeted to the TCR
Here we report on T cell development and TCR
Flow Cytometry.
PCR.
TCR
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During lymphocyte development immunoglobulin and TCR genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction 1. The V(D)J recombination reaction is mediated by the products of the lymphocyte specific recombination activating genes RAG1 and RAG2 which recognize and cleave recombination signal sequences located adjacent to the coding V, D, and J segments 2345. T and B lymphocyte development requires V(D)J recombination; in the absence of RAG1 and RAG2 67 or factors that repair the double strand DNA breaks created during V(D)J recombination there is a complete block in the early stages of B and T cell development 89101112131415. and CD3 components to produce the pre-TCR complex (for reviews, see references 17 and 18). Pre-TCR expression downregulates RAG expression and induces T cells to mature to the CD4+CD8+ double positive (DP) stage. Upon entering the DP stage there is a second wave of RAG expression and V(D)J recombination 192021. Regulation of RAG expression in developing thymocytes has been studied by two groups 2223. Transgenic experiments with large bacterial artificial chromosomes (BACs) that carry fluorescent protein indicator genes in place of the RAG genes, showed that a cis element 35–70 kb 5' of RAG2 is required for the second wave of RAG expression 22. However, RAG2–/– blastocyst reconstitution experiments indicated that T cell development could be rescued with as little as 9 kb of sequence upstream of RAG2 23. Thus, the cis requirements for RAG reinduction in DP thymocytes and the functional consequences of reinduction remain poorly defined. locus. The TCR/
locus is a 1 megabase locus that contains the locus nested between the V and the J segments; there are 61 J segments spread over 70 kb of DNA 242526. TCR recombination is believed to begin at the 5' end of the J cluster and progress to the 3' Js during thymocyte maturation 272829. This idea is indirectly supported by the finding of sterile transcripts emanating from the 5' T early promoter (TEA) in late DN thymocytes 3031. Successful rearrangement and expression of TCR genes is marked by an increase in cell surface CD3/TCR levels, but expression of a TCR/β dimer is not sufficient to turn off RAG expression and V(D)J recombination. TCR recombination and RAG expression persist until positive selection 3233. Continued TCR recombination in cells that express nonselected /β TCRs might result in absence of allelic exclusion, and could theoretically interfere with clonal selection 34. Persistent recombination may nonetheless be advantageous if nonselected or self-reactive receptors are replaced by useful receptors thereby salvaging thymocytes that would otherwise be deleted. Indeed, in transgenic and gene targeted mice, secondary TCR recombination efficiently replaces TCRs that cannot be positively selected 2033353637. Despite the potential importance of secondary TCR recombination for tolerance and repertoire diversification the extent to which secondary recombination contributes to the TCR repertoire in normal mice has not been determined. recombination in mice that are unable to upregulate RAG expression in DP thymocytes. The results indicate that secondary V(D)J recombination makes a major contribution to the normal TCR repertoire.
Materials and Methods
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Mice.
Clone m3e8A is an 80-kb yeast artificial chromosome (YAC) containing the RAG1 and RAG2 genes (RYAC), identified by screening the YAC library of St. Mary's Hospital Medical School 38 with primers specific for RAG1 (Genethon). YAC DNA was purified by pulsed field electrophoresis as described 39, and microinjected into the pronuclei of fertilized ova of RAG1–/– mice (129/SvxCD1 F1) 6. Transgenic founders (RYII and RYIII) were bred for seven generations to C57Bl/6 RAG1–/– mice (The Jackson Laboratory). The single-copy line RYII was also bred to RAG2–/– mice (Taconic Farms) 7. The RAG2–/– TCRβ mice were from Taconic Farms.
Antibodies used were: PE anti-CD25, biotin anti-CD44, fluorescein anti-CD3, PE–anti-HSA, PE anti-CD8, allophycocyanin (APC) anti-CD4, APC anti-CD8, PE anti-TCRβ, and APC anti-B220 (BD PharMingen). Biotinylated antibodies were visualized with streptavidin-RED613 (GIBCO BRL). Acquisition and analysis were performed with a FACSCaliburTM and CELLQuestTM (Becton Dickinson). Subpopulations of thymocytes and spleen cells were sorted using a FACS VantageTM (Becton Dickinson) and final purity was >98%.
For reverse transcription (RT)-PCR total RNA from 50,000 cells from thymus or spleen/lymph nodes was prepared with TRIzol, primed with oligo-dT, and reverse transcribed with Superscript II (GIBCO/BRL). RAG1 primers were 5'-CAACCAAGCTGCAGACATTCTAGCACTC-3' and 5'-CACGTCGATCCGGAAAATCCTGGCAATG-3'. β-actin primers were 5'-TACCACTGGCATCGTGATGGACT-3' and 5'-TTTCTGCATCCTGTCGGCAAT-3'. PCR was performed at 94°C 30 s, 62°C 60 s, 72°C 45 s, for 32 cycles. VJ genes were amplified from cDNA primed with the chain constant region–specific primer 5'-ATCCATAGCCTTCATGTCCA-3'. PCR was nested using a C primer 5'-TCAAAGTCGGTGAACAGGCA-3' and a degenerate V primer AGAAGGTGAAGCAGAGNNM as described 40 for two cycles at 94°C 30 s, 52°C 60 s, 72°C 60 s, followed by 40 cycles at 94°C 30 s, 55°C 60 s, 72°C 60 s. PCR products were cloned before sequencing. V-Jβ sequences were cloned and sequenced using the degenerate Vβ primers 5'-GGMCAYAVTGCTVTKTWCTGGTA-3', 5'- AAYCATGAYAMMATGTACTGGTA-3', 5'-CARGCHCCTTCGVTGDNYTGGTA-3', and Cβ nested primers, 5'- TCAGGCAGTAGCTATAATTGCTCTC-3', 5'-TTGCCATTCACCCACCAGCTC-3', and 5'-GCTCAGCTCCACGTGGTCAG-3'. (M = A/C, R = A/G, W = A/T, Y = C/T, K = G/T, V = A/C/G, H = A/C/T, D = A/G/T, N = A/C/G/T). J and Vβ-Jβ segments were identified by comparison to the GenBank/EMBL/DDBJ database sequences with accession nos. M64239 and AE000663–5, respectively. gene recombination was measured by PCR reactions using the degenerate V primer described above in conjunction with either a proximal J primer 5'-ACATGAGCTCACTGTCAGCT-3' (3' to J24.2) or a distal J primer 5'-TTACTTGGCTTCACTGTGAG-3' (3' to J57.9; see Fig. 4). PCR was at: 94°C 30 s, 55°C 60 s, 72°C 3 min for 25 cycles. PCR products were analyzed by electrophoresis in agarose and visualized by blotting and hybridizing with radiolabeled J probes 5'-AGGAGGGTCTGCGAAGCTCATCTTT-3' (proximal) and 5'-ACCAATACAGGCAAATTAACCTTTG-3' (distal). For single cell PCR, the stained cells and the cytometer sheath fluid were treated with 0.25 pg/ml RNase A to avoid contamination with RNA from lysed cells. Single CD25+CD44– DN T cells from RYIIRAG1–/– and wild-type mice were sorted into 96-well plates containing 4 µl catch buffer (75 mM NaCl, 1 mM DTT, 4 units Promega RNAsin, 7 units Eppendorf Prime RNase inhibitor) per well and placed on dry ice. RT reactions were performed by addition of 7 µl of random hexamer solution (300 ng random hexamers, 2 pmole RAG1-specific primer: 5'-CTTGAGTCCCCGATGGGCAGTAAA-3', 1.4% NP-40, 10 units Eppendorph Prime RNase inhibitor, water) followed by a 1-min incubation at 37°C followed by addition of 14 µl of RT reaction solution (5 µl of 5x first strand Superscript buffer, 1 mM dNTPs, 8 mM DTT, 14 units Promega RNAsin, 7 units Prime RNAsin, 0.5 µl Superscript II [GIBCO BRL]). RT was for 10 min at 25°C, followed by 30 min at 37°C, and the enzyme was destroyed by incubation at 90°C for 6 min. 2.5 µl (10%) of the cDNA for each cell was used for nested PCR reactions to amplify RAG1 cDNA or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a positive control. The primers for both RAG1 and GAPDH were designed to span introns to distinguish cDNA from genomic DNA. PCR primers: RAG1 external sense: 5'-GCTATCTCTGTGGCATCGAGTGTT-3'; RAG1 external antisense: 5'-AAA-GACTTTGGGTTTTAGC-3'; RAG1-nested-sense: 5'-GCCG-GGAGGCCTGTGGAG-3'; RAG1 nested antisense: 5'-CCG-TCGGGTGGATGGAGTCAA-3'; GAPDH external sense: 5'-GGTCATCATCTCCGCCCCTTCTG-3'; GAPDH exter-nal antisense: 5'-CACCCTGTTGCTGTAGCCGTATTC-3'; GAPDH nested sense: 5'-TTTGGCATTGTGGAAGGGCTCAT-3'; GAPDH nested antisense: 5'-TCGAAGGTGGAAG-AGTGGGAGTTG-3'. PCR cycle conditions were: 94°C 15 min, 40 cycles of 94°C 30 s, 55°C 30 s, 72°C 30 s, and finally a 7-min extension at 72°C. A total of 138 cells were assayed for RAG1 expression from the RYIIRAG1–/– mice and 64 cells were assayed from wild-type mice. To ensure that difference between RYIIRAG1–/– and control samples was not due to the fourfold decrease in RAG1 mRNA in RYIIRAG1–/– cells we performed four separate PCR experiments on each RYIIRAG1–/– cell and diluted control cell cDNA.
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19330.11, J42417.4, and J4.1 probes 28.
Immunofluorescence.
In situ staining for Nijmegen breakage syndrome (NBS)1 was as described previously 41.
Online Supplemental Data Section.
The online supplemental data contains two tables, online supplemental Tables S1 and S2. Online supplemental material is available at http://www.jem/org/cgi/content/full/194/4/471/DC1.
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To determine whether the difference in RAG expression between RYAC and wild-type mice affects TCRβ recombination and expression we cloned and characterized TCRβ mRNAs from thymus. We find no significant differences in TCRβ V, D, or J usage and no significant differences in the nature of the joints between RYAC and wild-type mice (online supplemental Table S2). We conclude that the pattern of RAG1 and RAG2 expression in DN thymocytes in RYAC mice resembles that found in BAC transgenic mice and that this level of expression is sufficient for TCRβ V(D)J recombination.
RAG Expression in DP Thymocytes.
To determine whether RYAC directs regulated RAG expression in DP thymocytes we purified these cells from RYIIRAG1–/–, RYIIIRAG1–/–, RYIIRAG2–/–, and wild-type control mice and measured steady-state levels of RAG1 and RAG2 mRNA by semiquantitative RT-PCR (Fig. 1 B). We found that there was little RAG1 or RAG2 expression in DP thymocytes in RYIIRAG1–/–, RYIIIRAG1–/–, and RYIIRAG2–/– mice (60–120-fold less than in wild-type mice in five experiments). Thus, RYII and RYIII transgenic mice resemble previously characterized BAC transgenic mice in that a cis element that is not contained in RYAC is required for regulated RAG1 and RAG2 expression in DP thymocytes.
T Cell Development.
To determine the physiologic consequences of loss of RAG1 and RAG2 expression in DP cells we analyzed T cell development in RYIIRAG1–/–, RYIIRAG2–/–, and RYIIIRAG1–/– mice. We found that the developmental profile of DN cells in RYIIRAG1–/–, RYIIRAG2–/–, and RYIIIRAG1–/– mice was similar to that of wild-type thymocytes but that the number of thymocytes in the most mature DN subset (CD25–CD44–) was decreased (Fig. 2 A). There was also a corresponding, small but consistent, increase in the percentage of CD25+ CD44– T cells (the immediate precursors of CD25– CD44–T cells; Fig. 2 A) and a fourfold decrease in the percentage of these cells in the S or G2/M phase of the cell cycle in RYIIRAG1–/– and RYIIIRAG1–/– mice (as measured by analysis of DNA content after DAPI staining; data not shown). The accumulation of nonproliferating CD25+ CD44– thymocytes in the transgenic mice resembles the accumulation of these cells in RAG1–/– mice and is consistent with variegated RAG expression in this stage of T cell development (see above).
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, CD3med cells express TCR but have not yet completed positive selection, and CD3high cells have completed selection and as a result upregulate surface TCR expression. RYIIRAG1–/–, RYIIRAG2–/–, and RYIIIRAG1–/– mice showed an altered distribution of CD3 expression: the majority of thymocytes in these mice were CD3low whereas this is only a minor population in the wild-type thymus (Fig. 2 D). In addition, we found that the transgenic mice displayed a two- to fourfold decrease in the number of CD3high cells, which is also consistent with the decrease in the number of SP thymocytes (Fig. 2 C, five experiments). Similar results were obtained by anti-TCRβ staining (not shown). We conclude that decreased RAG expression in DP thymocytes in RYIIRAG1–/–, RYIIRAG2–/–, and RYIIIRAG1–/– mice leads to a decrease in the number of thymocytes expressing medium and high levels of TCR.
TCR Recombination.
The relative decrease in RAG expression in DP thymocytes and the increase in the percentage of CD3low cells in RYAC mice suggest that there might be lower levels of TCR recombination. Double stranded DNA break intermediates created during V(D)J recombination at the TCR locus can be visualized in developing thymocytes by staining nuclei with antibodies to the NBS1 protein 41. To examine the extent of V(D)J recombination in RYIIRAG1–/– thymocytes directly, we stained purified DP cells from these mice with antibodies to NBS1 and compared them to RAG2–/– TCRβ transgenic and wild-type mice (Fig. 3). In agreement with previous results, 25% of wild-type DP thymocytes had NBS1 foci. These foci were not detectable in RAG2–/– TCRβ mice, which have normal number of DP cells but do not undergo V(D)J recombination 21. In contrast, 5% of the DP thymocytes from RYIIRAG1–/– mice showed NBS1 foci. Thus, the percentage of DP thymocytes with double stranded breaks in RYIIRAG1–/– mice is decreased in a manner consistent with impaired TCR recombination.
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genes expressed in RYAC transgenic mice differ from the wild-type controls we amplified and sequenced TCR mRNAs from thymus and spleen (Fig. 4 A). Although the V genes expressed in RYIIRAG1–/– mice and the VJ junctions were indistinguishable from controls, the Js used in these mice were highly biased to the proximal end of the J cluster (Fig. 4 A, and not shown). Whereas only 8% of the wild-type TCR genes in the thymus used a J from the proximal 10 kb of this cluster, 41% of the TCR expressed in the RYIIRAG1–/– thymus used these 5' most proximal Js (42; Fig. 4 A). Further, in wild-type mice 42% of the Js were from the distal half of the locus whereas only 7% of the TCR genes expressed in RYIIRAG1–/– mice carry Js from the distal part of the locus (42; Fig. 4 A). Proximal skewing of the Js was even more evident in T cells that had undergone selection and been exported to the spleen. In the wild-type, 75% of spleen T cells expressed TCRs using distal Js whereas only 3% of the TCRs cloned from RYII spleen T cells used distal Js (Fig. 4 A). We conclude that the TCR genes expressed in RYIIRAG1–/– mice are highly biased toward proximal J usage.
To determine whether skewed J usage is due to biased TCR recombination we measured recombination by PCR using primers specific for the 5' and 3' ends of the J locus. Both V to 5' J and V to 3' J rearrangements were readily detected in wild-type mice but only V to 5' J rearrangements were found in RYIIRAG1–/– and RYIIRAG2–/– mice (Fig. 4 B). Thus, there appears to be a relative absence of recombination to the 3' J locus in RYIIRAG1–/– and RYIIRAG2–/– mice. We used Southern blotting on DNA purified from CD3low, med, high thymocytes with probes that hybridize to the 5', middle, and 3' ends of the J cluster to measure TCR recombination directly 28. The amount of recombination was standardized with a C probe and quantified by phosphorimager analysis (Fig. 5). Overall, RYIIRAG1–/– and RYIIIRAG1–/– mice showed less J recombination than wild-type controls and almost all of the recombination was restricted to the 5' portion of the J cluster (Fig. 5).
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gene to become CD3med; therefore the theoretical minimum V(D)J recombination that would allow a T cell to become CD3med is 50%. In wild-type mice J recombination was detected on 78–80% of the chromosomes in T cells reaching this stage in development, indicating that most CD3med cells have undergone more than a single V(D)J rearrangement (34434445; Fig. 5). In contrast, CD3med thymocytes in RYIIRAG1–/– and RYIIIRAG1–/– mice showed only 58 and 57% 5' J recombination. Thus, most CD3med cells in RYIIRAG1–/– and RYIIIRAG1–/– mice have only attempted V(D)J recombination on one chromosome.
In wild-type mice TCR recombination may begin with recombination to 5' Js, and 3' J recombination seems to increase as thymocytes progress to more mature stages in development 28. For example, there was 70% 5', 29% middle, and 14% 3' J recombination in CD3low DP cells and this increased to 83, 51, and 19%, respectively, in SP cells in wild-type mice (28; Fig. 5, bottom). RYIIRAG1–/– and RYIIIRAG1–/– mice showed a more drastic bias to 5' J recombination in CD3low DP thymocytes and there was no significant additional recombination to the middle and 3' part of the locus as thymocytes progressed in development (Fig. 5). We conclude that RYIIRAG1–/– and RYIIIRAG1–/– thymocytes differ from wild-type in that they do not recombine 3' Js in the DP compartment.
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In vitro analysis of cis regulation of the RAG1 promoter revealed only nonspecific basal promoter activity 5455565758. In contrast, the RAG2 promoter displayed preferential activity in lymphoid cell lines that was PAX5 and GATA3 dependent but the activity was not developmentally restricted 5960. Two systems have been used to study RAG transcription in vivo: RAG2–/– blastocyst reconstitution and transgenic reporters 2223. In the RAG2–/– blastocyst reconstitution experiments an 18-kb genomic fragment extending from 9 kb upstream of the RAG2 promoter to 2.4 kb downstream of the 3' UTR was able to reconstitute T cell development 23. These experiments suggested that all of the information required for RAG regulation might be found proximal to RAG2 23. In contrast, the transgenic reporter experiments showed that an element in the genomic region 35–70 kb upstream of the RAG2 promoter is required for RAG1 and RAG2 expression in DP T cells 22. Our results with RYAC confirm the presence of a distal regulatory element that regulates both RAG1 and RAG2 reinduction and also reconcile the apparent discrepancies between the two sets of in vivo experiments. The RYAC, which has only 12 kb of genomic sequence 5' of the RAG2 promoter, resembles the 18-kb fragment used in the RAG2–/– blastocyst system in that both DNA fragments reconstitute the T cell compartment. However, T cell reconstitution by RYAC occurs in absence of the second wave of RAG expression in DP T cells. Thus, the distal element identified in the transgenic reporter system could not have been detected by the T cell rescue experiments in the RAG2–/– blastocyst system.
TCR Recombination.
In wild-type mice, TCR recombination is thought to proceed coordinately on both chromosomes without allelic exclusion 3443444561. Only a small fraction of the TCR genes in CD3med DP T cells are in the germline configuration, and there is an increase in the amount of 3' J recombination as thymocytes mature, which is consistent with continuing chromosome loss due to continued TCR recombination in the DP stage 2729. In contrast, in RYAC mice, almost half of the TCR genes in CD3med DP T cells are in the germline configuration; only 57% of the TCR alleles are recombined. All CD3med DP T cells must express a TCR gene, therefore 57% recombination is just over the theoretical limit of 50% required for TCR expression in these cells. This indicates that most CD3med transgenic T cells undergo TCR rearrangement on only one allele and suggests that TCR recombination begins on one chromosome.
The 5' portion of the TCR locus is believed to be the first part of the J cluster to become available for recombination 272829. Sterile transcription from the TEA is associated with accessibility to this part of the locus in the late DN stage of T cell development, and in the absence of the TEA the 5' most Js are not recombined 3031. The 3' portion of the J cluster is thought to become accessible for recombination later in T cell development in a TEA-independent but TCR enhancer–dependent fashion 26626364. Thus, the 5' J recombination we find in RYAC transgenic mice might be accounted for by residual RAG protein in thymocytes transiting from the DN to the DP stage and the absence of 3' J recombination due to the relative lack of RAG expression in DP thymocytes. Alternatively, the 1–2% of normal levels of RAG expression we find in DP T cells could be enough to recombine only 5' J genes. In either case, the finding that only the 5' J genes are rearranged in the absence of the normal second wave of RAG expression clearly demonstrates that TCR recombination is ordered, and that the 5' side of the J cluster is the first to become accessible to the recombinase 27282930.
The second wave of RAG expression in DP thymocytes is normally terminated during positive selection 20323365. Transgenic and gene targeted mice that carry nonselecting receptors undergo persistent TCR locus secondary recombination 333637. Additional support for the idea that there is continuing recombination in DP thymocytes comes from the finding that 3' J rearrangements accumulate as normal thymocytes progress to the mature SP stage 2837. RYAC transgenic thymocytes fail to activate the second wave of RAG expression and fail to accumulate 3' J rearrangements that are indicative of secondary recombination. Despite this 5' bias and apparent absence of continued V(D)J recombination, mature RYAC thymocytes show higher levels of J chromosome loss than immature CD3low DP thymocytes. We cannot rule out the possibility that the 1–2% of normal level RAG expression in RYAC DP thymocytes targets continuing 5' J but not 3' J recombination, but this seems unlikely. It seems more likely that the increase in chromosome loss in mature T cells in RYAC mice simply reflects selection for the few T cells that have randomly recombined both TCR alleles because these cells have a higher probability of producing an in frame TCR gene.
By comparing TCR recombination in wild-type mice with RYAC transgenic mice that do not reinduce RAG expression, we can estimate the contribution of secondary TCR recombination to the normal /β T cell repertoire. Less than 10% of all TCR genes expressed in mature RYAC T cells contain Js from the 3' half of the J cluster. In contrast, 75% of all TCR genes expressed by splenic T cells in wild-type mice carry Js from the 3' half of the J cluster. Thus, at least 65% of the TCR genes in wild-type mice appear to be products of secondary recombination, a much higher level than the 25% estimated for Ig
receptor editing in B cells 66. We conclude that the majority of the TCR repertoire in the spleen of wild-type mice is the product of secondary V(D)J recombination and that secondary recombination makes a major contribution to the normal /β TCR repertoire.
It has been proposed that 
/ T cells are the evolutionary precursors of β T cells 67. The V(D)J recombination in RYAC /β T cells is developmentally similar to that of the / cells in that it is mostly absent in the DP compartment. Unlike / T cells, in which the rearrangement is not ordered and shows no allelic exclusion 68, the simple pattern of rearrangement in RYAC /β T cells results in virtual allelic exclusion of the TCR locus. It leads, however, to a two- to threefold decrease in the number of SP thymocytes and a substantial decrease in the complexity of the /β T cell receptor repertoire, as only a fraction of the available Js are used. We speculate that the cis regulatory element that induces the second wave of RAG expression is a late addition to the RAG locus that was selected in evolution at the expense of allelic exclusion because this element increased the diversity of the repertoire.
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Acknowledgments |
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This work was supported in part by grants from National Institutes of Health to M.C. Nussenzweig. M.C. Nussenzweig is a Howard Hughes Medical Institute investigator.
Submitted: 11 May 2001
Revised: 18 June 2001
Accepted: 11 July 2001
Abbreviations used in this paper: APC, allophycocyanin; BAC, bacterial artificial chromosome; DN, double negative; DP, double positive; NBS, Nijmegen breakage syndrome; SP, single positive; RAG, recombination activating gene; RT, reverse transcription; TEA, T early
promoter; YAC, yeast artificial chromosome.
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References |
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Tonegawa S.. Somatic generation of antibody diversity, Nature., 302, 1983, 575–581.[Medline]
Fugmann S.D., Lee A.I., Shockett P.E., Villey I.J. & Schatz D.G.. The RAG proteins and V(D)J recombinationcomplexes, ends, and transposition, Annu. Rev. Immunol., 18, 2000, 495–527.[Medline]
McBlane J.F., van Gent D.C., Ramsden D.A., Romeo C., Cuomo C.A., Gellert M. & Oettinger M.A.. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps, Cell., 83, 1995, 387–395.[Medline]
Oettinger M.A., Schatz D.G., Gorka C. & Baltimore D.. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination, Science., 248, 1990, 1517–1523.
Schatz D.G., Oettinger M.A. & Baltimore D.. The V(D)J recombination activating gene, RAG-1, Cell., 59, 1989, 1035–1048.[Medline]
Mombaerts P., Iacomini J., Johnson R.S., Herrup K., Tonegawa S. & Papaioannou V.E.. RAG-1-deficient mice have no mature B and T lymphocytes, Cell., 68, 1992, 869–877.[Medline]
Shinkai Y., Rathbun G., Lam K.P., Oltz E.M., Stewart V., Mendelsohn M., Charron J., Datta M., Young F., Stall A.M. & Alt F.W.. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement, Cell., 68, 1992, 855–867.[Medline]
Barnes D.E., Stamp G., Rosewell I., Denzel A. & Lindahl T.. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice, Curr. Biol., 8, 1998, 1395–1398.[Medline]
Difilippantonio M.J., Zhu J., Chen H.T., Meffre E., Nussenzweig M.C., Max E.E., Ried T. & Nussenzweig A.. DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation, Nature., 404, 2000, 510–514.[Medline]
Frank K.M., Sekiguchi J.M., Seidl K.J., Swat W., Rathbun G.A., Cheng H.L., Davidson L., Kangaloo L. & Alt F.W.. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV, Nature., 396, 1998, 173–177.[Medline]
Gao Y., Sun Y., Frank K.M., Dikkes P., Fujiwara Y., Seidl K.J., Sekiguchi J.M., Rathbun G.A., Swat W. & Wang J.. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis, Cell., 95, 1998, 891–902.[Medline]
Gu Y., Seidl K.J., Rathbun G.A., Zhu C., Manis J.P., van der Stoep N., Davidson L., Cheng H.L., Sekiguchi J.M. & Frank K.. Growth retardation and leaky SCID phenotype of Ku70-deficient mice, Immunity., 7, 1997, 653–665.[Medline]
Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M.C. & Li G.C.. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination, Nature., 382, 1996, 551–555.[Medline]
Ouyang H., Nussenzweig A., Kurimasa A., Soares V.d.C., Li X., Cordon-Cardo C., Li W.-h., Cheong N., Nussenzweig M. & Iliakis G.. Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination in vivo, J. Exp. Med., 186, 1997, 921–929.
Zhu C., Bogue M.A., Lim D.S., Hasty P. & Roth D.B.. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates, Cell., 86, 1996, 379–389.[Medline]
Godfrey D., Kennedy J., Mombaerts P., Tonegawa S. & Zlotnik A.. Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3–CD4–CD8– thymocyte differentiation, J. Immunol., 152, 1994, 4783–4792.[Abstract]
von Boehmer H. & Fehling H.J.. Structure and function of the pre-T cell receptor, Annu. Rev. Immunol., 15, 1997, 433–452.[Medline]
Kruisbeek A.M., Haks M.C., Carleton M., Wiest D.L., Michie A.M. & Zuniga-Pflucker J.C.. Branching out to gain controlhow the pre-TCR is linked to multiple functions, Immunol. Today., 21, 2000, 637–644.[Medline]
Wilson A., Held W. & MacDonald H.. Two waves of recombinase gene expression in developing thymocytes, J. Exp. Med., 179, 1994, 1355–1360.
Petrie H., Livak F., Schatz D., Strasser A., Crispe I. & Shortman K.. Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes, J. Exp. Med., 178, 1993, 615–622.
Shinkai Y., Koyasu S., Nakayama K., Murphy K.M., Loh D.Y., Reinherz E.L. & Alt F.W.. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes, Science., 259, 1993, 822–825.
Yu W., Misulovin Z., Suh H., Hardy R.R., Jankovic M., Yannoutsos N. & Nussenzweig M.C.. Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2, Science., 285, 1999, 1080–1084.
Monroe R.J., Chen F., Ferrini R., Davidson L. & Alt F.W.. RAG2 is regulated differentially in B and T cells by elements 5' of the promoter, Proc. Natl. Acad. Sci. USA., 96, 1999, 12713–12718.
Jouvin-Marche E., Hue I., Marche P.N., Liebe-Gris C., Marolleau J.P., Malissen B., Cazenave P.A. & Malissen M.. Genomic organization of the mouse T cell receptor V alpha family, EMBO J., 9, 1990, 2141–2150.[Medline]
Koop B.F., Wilson R.K., Wang K., Vernooij B., Zallwer D., Kuo C.L., Seto D., Toda M. & Hood L.. Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region, Genomics., 13, 1992, 1209–1230.[Medline]
Krangel M.S., Hernandez-Munain C., Lauzurica P., McMurry M., Roberts L.J. & Zhong X.-P.. Developmental regulation of the V(D)J recombination at the TCR/ locus, Immunol. Rev., 165, 1998, 131–147.[Medline]
Roth M.E., Holman P.O. & Kranz D.M.. Nonrandom use of J alpha gene segments. Influence of V alpha and J alpha gene location, J. Immunol., 147, 1991, 1075–1081.[Abstract]
Petrie H., Livak F., Burtrum D. & Mazel S.. T cell receptor gene recombination patterns and mechanismscell death, rescue, and T cell production, J. Exp. Med., 182, 1995, 121–127.
Thompson S.D., Pelkonen J. & Hurwitz J.L.. First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus, J. Immunol., 145, 1990, 2347–2352.[Abstract]
Wilson A., de Villartay J.P. & MacDonald H.R.. T cell receptor delta gene rearrangement and T early alpha (TEA) expression in immature alpha beta lineage thymocytesimplications for alpha beta/gamma delta lineage commitment, Immunity., 4, 1996, 37–45.[Medline]
Villey I., Caillol D., Selz F., Ferrier P. & de Villartay J.P.. Defect in rearrangement of the most 5' TCR-J alpha following targeted deletion of T early alpha (TEA)implications for TCR alpha locus accessibility, Immunity., 5, 1996, 331–342.[Medline]
Turka L.A., Schatz D.G., Oettinger M.A., Chun J.J., Gorka C., Lee K., McCormack W.T. & Thompson C.B.. Thymocyte expression of RAG-1 and RAG-2termination by T cell receptor cross-linking, Science., 16, 1991, 778–781.
Borgulya P., Kishi H., Uematsu Y. & von Boehmer H.. Exclusion and inclusion of and β T cell receptor alleles, Cell., 69, 1992, 529–537.[Medline]
Malissen M., Trucy J.J., Jouvin-Marche E., Cazenave P., Scollay R. & Malissen B.. Regulation of TCR and β gene allelic exclusion during T-cell development, Immunol. Today., 13, 1992, 315–322.[Medline]
Malissen M., Trucy J., Letourneur F., Rebai N., Dunn D.E., Fitch F.W., Hood L. & Malissen B.. A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition, Cell., 55, 1988, 49–59.[Medline]
McGargill M.A., Derbinski J.M. & Hogquist K.A.. Receptor editing in developing T cells, Nat. Immunol., 1, 2000, 336–341.[Medline]
Wang F., Huang C.-Y. & Kanagawa O.. Rapid deletion of rearranged T cell antigen receptor (TCR) Valpha-Jalpha segment by secondary rearrangement in the thymusrole of continuous rearrangement of TCR alpha chain gene and positive selection in the T cell repertoire formation, Proc. Natl. Acad. Sci. USA., 95, 1998, 11834–11839.
Chartier F.L., Keer J.T., Sutcliffe M.J., Henriques D.A., Mileham P. & Brown S.D.. Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast, Nat. Genet., 1, 1992, 132–136.[Medline]
Yannoutsos N., Ijzermans J.N., Harkes C., Bonthuis F., Zhou C.Y., White D., Marquet R.L. & Grosveld F.. A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection, Genes Cells., 1, 1996, 409–419.[Abstract]
Broeren C.P.M., Goerge G.M., Verjans G.M., Van Eden W., Kusters J.G., Lenstra J.A. & Longtenberg T.. Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification, Eur. J. Immunol., 21, 1991, 569–575.[Medline]
Chen H.T., Bhandoola A., Difilippantonio M.J., Zhu J., Brown M.J., Tai X., Rogakou E.P., Brotz T.M., Bonner W.M., Ried T. & Nussenzweig A.. Response to RAG-mediated V(D)J cleavage by NBS1 and -H2AX, Science., 290, 2000, 1962–1964.
Riegert P. & Gilfillan S.. A conserved sequence block in the murine and human TCR Ja regionassessment of regulatory function in vivo, J. Immunol., 162, 1999, 3471–3480.
Davis M.M. & Bjorkman P.J.. T-cell antigen receptor genes and T-cell recognition, Nature., 334, 1988, 395–402.[Medline]
Pircher H., Michalopoulos E.E., Iwamoto A., Ohashi P.S., Baenziger J., Hengartner H., Zinkernagel R.M. & Mak T.W.. Molecular analysis of the antigen receptor of virus-specific cytotoxic T cells and identification of a new V alpha family, Eur. J. Immunol., 17, 1987, 1843–1846.[Medline]
Uematsu Y.. Preferential association of alpha and beta chains of the T cell antigen receptor, Eur. J. Immunol., 22, 1992, 603–606.[Medline]
van Gent D.C., Mizuuchi K. & Gellert M.. Similarities between initiation of V(D)J recombination and retroviral integration, Science., 271, 1996, 1592–1594.[Abstract]
Landree M.A., Wibbenmeyer J.A. & Roth D.B.. Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination, Genes Dev., 13, 1999, 3059–3069.
Kim D.R., Dai Y., Mundy C.L., Yang W. & Oettinger M.A.. Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase, Genes Dev., 13, 1999, 3070–3080.
Kennedy A.K., Guhathakurta A., Kleckner N. & Haniford D.B.. Tn10 transposition via a DNA hairpin intermediate, Cell., 95, 1998, 125–134.[Medline]
Hiom K., Melek M. & Gellert M.. DNA transposition by the RAG1 and RAG2 proteinsa possible source of oncogenic translocations, Cell., 94, 1998, 463–470.[Medline]
Besmer E., Mansilla-Soto J., Cassard S., Sawchuk D.J., Brown G., Sadofsky M., Lewis S.M., Nussenzweig M.C. & Cortes P.. Hairpin coding end opening is mediated by RAG1 and RAG2 proteins, Mol. Cell., 2, 1998, 817–828.[Medline]
Agrawal A., Eastman Q.M. & Schatz D.G.. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system, Nature., 394, 1998, 744–751.[Medline]
Shockett P.E. & Schatz D.G.. DNA hairpin opening mediated by the RAG1 and RAG2 proteins, Mol. Cell. Biol., 19, 1999, 4159–4166.
Zarrin A.A., Fong I., Malkin L., Marsden P.A. & Berinstein N.L.. Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions, J. Immunol., 159, 1997, 4382–4394.[Abstract]
Kurioka H., Kishi H., Isshiki H., Tagoh H., Mori K., Kitagawa T., Nagata T., Dohi K. & Muraguchi A.. Isolation and characterization of a TATA-less promoter for the human RAG-1 gene, Mol. Immunol., 33, 1996, 1059–1066.[Medline]
Kitagawa T., Mori K., Kishi H., Tagoh H., Nagata T., Kurioka H. & Muraguchi A.. Chromatin structure and transcriptional regulation of human RAG-1 gene, Blood., 88, 1996, 3785–3791.
Fuller K. & Storb U.. Identification and characterization of the murine Rag1 promoter, Mol. Immunol., 34, 1997, 939–954.[Medline]
Brown S.T., Miranda G.A., Galic Z., Hartman I.Z., Lyon C.J. & Aguilera R.J.. Regulation of the RAG-1 promoter by the NF-Y transcription factor, J. Immunol., 158, 1997, 5071–5074.[Abstract]
Lauring J. & Schlissel M.S.. Distinct factors regulate the murine RAG-2 promoter in B- and T-cell lines, Mol. Cell. Biol., 19, 1999, 2601–2612.
Kishi H., Wei X.C., Jin Z.X., Fujishiro Y., Nagata T., Matsuda T. & Muraguchi A.. Lineage-specific regulation of the murine RAG-2 promoterGATA-3 in T cells and Pax-5 in B cells, Blood., 95, 2000, 3845–3852.
Casanova J., Romero P., Widmann C., Kourilsky P. & Maryanski J.. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptideimplications for T cell allelic exclusion and antigen-specific repertoire, J. Exp. Med., 174, 1991, 1371–1383.
Hernandez-Munain C., Sleckman B.P. & Krangel M.S.. A developmental switch from TCR delta enhancer to TCR alpha enhancer function during thymocyte maturation, Immunity., 10, 1999, 723–733.[Medline]
McMurry M.T. & Krangel M.S.. A role for histone acetylation in the developmental regulation of V(D)J recombination, Science., 287, 2000, 495–498.
Sleckman B.P., Bardon C.G., Ferrini R., Davidson L. & Alt F.W.. Function of the TCR alpha enhancer in alphabeta and gammadelta T cells, Immunity., 7, 1997, 505–515.[Medline]
Brandle D., Muller S., Muller C., Hengartner H. & Pircher H.. Regulation of RAG-1 and CD69 expression in the thymus during positive and negative selection, Eur. J. Immunol., 24, 1994, 145–151.[Medline]
Casellas R., Shih T.Y., Jasna R., Kleinewietfeld M., Nemazee D., Rajewsky K. & Nussenzweig M.C.. Contribution of receptor editing to the antibody repertoire, Science., 291, 2001, 1541–1544.
Flajnik M.F.. Churchill and the immune system of ectothermic vertebrates, Immunol. Rev., 166, 1998, 5–14.[Medline]
Sleckman B.P., Khor B., Monroe R. & Alt F.W.. Assembly of productive T cell receptor delta variable region genes exhibits allelic inclusion, J. Exp. Med., 188, 1998, 1465–1471.
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60 ng of template DNA diluted as shown. PCR on CD14 was used as loading control. Results are representative of two separate experiments with individual mouse samples.
, or GAPDH were used as loading controls for normalizing mRNA. The data shown are representative of five experiments. cDNA dilutions are indicated. WT, wild-type.
