Correspondence to: Nikos Yannoutsos, The Rockefeller University, Box 220, 1230 York Ave., New York, NY 10021. Tel:212-327-8068 Fax:212-327-8370 E-mail:yannoun{at}mail.rockefeller.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assembly of T cell receptor (TCR)/ß genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRß recombination in CD4-CD8-CD25+ thymocytes. In these cells, TCRß expression leads to clonal expansion, RAG downregulation, and TCRß 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
/ß 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 5070% 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
/ß 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
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (2) (3) (4) (5). T and B lymphocyte development requires V(D)J recombination; in the absence of RAG1 and RAG2 (6) (7) 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 (8) (9) (10) (11) (12) (13) (14) (15).
In thymocytes, V(D)J recombination is initiated at the TCRß locus in CD4-CD8- double negative (DN) T cells (16). Once a TCRß chain is expressed it combines with pre-T 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 (19) (20) (21). Regulation of RAG expression in developing thymocytes has been studied by two groups (22) (23). 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 3570 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.
In DP thymocytes V(D)J recombination is targeted to the TCR 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 (24) (25) (26). TCR
recombination is believed to begin at the 5' end of the J
cluster and progress to the 3' J
s during thymocyte maturation (27) (28) (29). This idea is indirectly supported by the finding of sterile transcripts emanating from the 5' T early
promoter (TEA) in late DN thymocytes (30) (31). Successful rearrangement and expression of TCR
genes is marked by an increase in cell surface CD3/TCR levels, but expression of a TCR
/ß dimer is not sufficient to turn off RAG expression and V(D)J recombination. TCR
recombination and RAG expression persist until positive selection (32) (33). Continued TCR
recombination in cells that express nonselected
/ß 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 (20) (33) (35) (36) (37). 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.
Here we report on T cell development and TCR 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-/- TCRß mice were from Taconic Farms.
Flow Cytometry.
Antibodies used were: PE anti-CD25, biotin anti-CD44, fluorescein anti-CD3, PEanti-HSA, PE anti-CD8, allophycocyanin (APC) anti-CD4, APC anti-CD8, PE anti-TCRß, 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%.
PCR.
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'. ß-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 regionspecific 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-Jß sequences were cloned and sequenced using the degenerate Vß primers 5'-GGMCAYAVTGCTVTKTWCTGGTA-3', 5'- AAYCATGAYAMMATGTACTGGTA-3', 5'-CARGCHCCTTCGVTGDNYTGGTA-3', and Cß 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 Vß-Jß segments were identified by comparison to the GenBank/EMBL/DDBJ database sequences with accession nos.
M64239 and
AE0006635, respectively.
TCR 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 J
24.2) or a distal J
primer 5'-TTACTTGGCTTCACTGTGAG-3' (3' to J
57.9; 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.
|
|
|
|
Quantitative Southern Blotting.
Quantitative Southern blotting was performed exactly as described using the J19330.11, J
42417.4, and J
4.1 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To clarify cis regulation of RAG expression in vivo we analyzed transgenic mice carrying an 80-kb YAC containing 33 kb of genomic sequence 5' of RAG1 and 12 kb of sequence 5' of RAG2 (RYAC; Fig 1). The two lines reported here (RYII single-copy and RYIII three-copy) were maintained by breeding to RAG1-/- or RAG2-/- mice and are referred to as RYIIRAG1-/-, RYIIRAG2-/-, and RYIIIRAG1-/-.
RAG Expression in DN Thymocytes.
To determine whether the RYAC directs expression of RAG1 and RAG2 in vivo we measured RAG1 mRNA levels in CD25+ DN thymocytes isolated from wild-type, RYIIRAG1-/-, RYIIIRAG1-/-, and RYIIRAG2-/- mice by flow cytometry. Steady-state levels of RAG1 and RAG2 mRNA were estimated by semiquantitative RT-PCR (Fig 1). CD25+ DN cells isolated from RYIIRAG1-/-, RYIIIRAG1-/-, and RYIIRAG2-/- mice expressed RAG mRNA at three- to fourfold lower levels than wild-type CD25+ DN cells (Fig 1 B). RAG indicator expression is variegated in mice that carry BAC transgenes similar to RYAC with a variable fraction of developing lymphocytes expressing the indicator transgene (22). To determine whether only a fraction of the DN cells in RYIIRAG1-/- and RYIIIRAG1-/- mice express RAG1 we conducted single-cell PCR experiments on purified CD44-CD25+ DN thymocytes. We found that the percentage of CD44-CD25+ thymocytes expressing RAG1 in RYIIRAG1-/- was threefold lower than in wild-type mice (online supplemental Table S1). This heterogeneity in gene expression is consistent with variegation seen in BAC reporter mice that carry similar transgenes (22).
To determine whether the difference in RAG expression between RYAC and wild-type mice affects TCRß recombination and expression we cloned and characterized TCRß mRNAs from thymus. We find no significant differences in TCRß 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 TCRß 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 (60120-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).
Despite the 9899% reduction in RAG expression in DP thymocytes, T cell development in RAG1-/- and RAG2-/- mice appeared to be reconstituted by the RYAC transgene as measured by the number of cells in the thymus and spleen. In addition, CD4 and CD8 staining profiles in the thymus and the periphery were similar for RYIIRAG1-/-, RYIIRAG2-/-, or RYIIIRAG1-/- transgenic mice and wild-type controls (Fig 2B and Fig C). However, the number of CD4+CD8- and CD4-CD8+ single positive (SP) cells was decreased two- to fourfold in the thymus of RYIIRAG1-/-, RYIIRAG2-/-, and RYIIIRAG1-/- mice (Fig 2 C). We conclude that T cell development can proceed to the SP stage with only minimal RAG mRNA expression in the DP compartment but fewer SP cells are produced. To examine TCR expression in developing T cells in RYIIRAG1-/-, RYIIRAG2-/-, and RYIIIRAG1-/- mice we stained thymocytes for expression of the TCRCD3 complex. Thymocytes can be divided into three groups based on low, medium, and high levels of CD3 expression. CD3low cells are the earliest DP cells and express TCRß but not TCR, 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-TCRß 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-/- TCRß 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-/- TCRß 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.
To determine whether the TCR 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 J
s 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 J
s ((42); Fig 4 A). Further, in wild-type mice 42% of the J
s were from the distal half of the locus whereas only 7% of the TCR
genes expressed in RYIIRAG1-/- mice carry J
s from the distal part of the locus ((42); Fig 4 A). Proximal skewing of the J
s 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 TCR
s using distal J
s whereas only 3% of the TCR
s cloned from RYII spleen T cells used distal J
s (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).
|
Thymocytes must rearrange and express at least one TCR 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 7880% 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 ((34) (43) (44) (45); 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' J
s, 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' J
s in the DP compartment.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RAG Regulation.
RAG1 and RAG2 are closely linked genes that are believed to originate from a transposon which entered the vertebrate lineage at the time of the evolution of jawed fish (2) (4) (5) (46) (47) (48) (49) (50) (51) (52) (53). Expression of the RAG nuclease is highly restricted, but the regulation of RAG expression remains poorly defined.
In vitro analysis of cis regulation of the RAG1 promoter revealed only nonspecific basal promoter activity (54) (55) (56) (57) (58). 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 (59) (60). Two systems have been used to study RAG transcription in vivo: RAG2-/- blastocyst reconstitution and transgenic reporters (22) (23). 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 3570 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 (34) (43) (44) (45) (61). 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 (27) (29). 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 (27) (28) (29). 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 J
s are not recombined (30) (31). 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
enhancerdependent fashion (26) (62) (63) (64). 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 12% 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 (27) (28) (29) (30).
The second wave of RAG expression in DP thymocytes is normally terminated during positive selection (20) (32) (33) (65). Transgenic and gene targeted mice that carry nonselecting receptors undergo persistent TCR locus secondary recombination (33) (36) (37). 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 (28) (37). 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 12% 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
/ß T cell repertoire. Less than 10% of all TCR
genes expressed in mature RYAC T cells contain J
s 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 J
s 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
/ß TCR repertoire.
It has been proposed that /
T cells are the evolutionary precursors of
ß T cells (67). The V(D)J recombination in RYAC
/ß 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
/ß 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
/ß T cell receptor repertoire, as only a fraction of the available J
s 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.
![]() |
Footnotes |
---|
The online version of this article contains supplemental material.
* 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.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This paper is dedicated to the memory of our friend Eugenia Spanopoulou, who provided the panel of YAC clones with the RAG1 and RAG2 genes. We thank Frank Isdell and Michelle Genova for the FACS® sorting, and the members of the Nussenzweig laboratory, as well as Dr. Harald von Boehmer, Dr. A. Tarakhovsky, and Dr. M. Shlomchik for useful discussions.
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
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|