Chromosomal excision of TCR
chain genes is dispensable for
ß T cell lineage commitment
Bernard Khor,
Tara D. Wehrly and
Barry P. Sleckman
Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St Louis, MO 63110, USA
Correspondence to: B. P. Sleckman; E-mail: sleckman{at}immunology.wustl.edu
 |
Abstract
|
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TCRß,
and
chain genes are assembled and expressed in double-negative thymocytes prior to
ß or 
T cell lineage commitment. Thus, cells committed to the
ß T cell lineage can possess completely assembled TCR
and/or TCR
chain genes. However, these genes are not expressed. TCR
chain gene expression may be silenced through the activity of a cis-acting silencer element. In the TCR
/
locus, the TCR
genes lie between the V
and J
gene segments, which rearrange by deletion. Moreover, V
to J
rearrangements occur on both alleles in essentially all developing
ß T cells. Consequently, both TCR
chain genes are excised from the chromosome and placed on extrachromosomal circles in mature
ß T cells. It has been proposed that this excision process is important for silencing TCR
gene expression and permitting
ß T cell lineage commitment. A gene-targeting CreloxP strategy was used to invert a 75-kb region of the TCR
/
locus encompassing all the J
gene segments, generating the TCR
/
I allele. Initial V
to J
rearrangements on the TCR
/
I allele occur by inversion, resulting in chromosomal retention of TCR
chain genes. These TCR
chain genes can be productively rearranged and are expressed at levels similar to TCR
chain genes in 
T cells. However,
ß T cell development appears unperturbed in TCR
/
I/I mice. Thus, excision of TCR
genes from the chromosome per se is not required for commitment of developing lymphocytes to the
ß T cell lineage.
Keywords: antigen receptor, development, T lymphocyte, V(D)J recombination
 |
Introduction
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T lymphocytes can be divided into two lineages based on expression of either
ß or 
TCRs. These cells develop from a common thymic precursor, the CD4/CD8 (double negative, DN) thymocyte. The molecular events that promote the differentiation of DN thymocytes along the
ß or 
T cell lineage pathway, although incompletely defined, require signals generated through the expression of T cell antigen receptor chains. Assembly and expression of TCRß,
and
chain genes occur in DN thymocytes, whereas assembly of TCR
chain genes is delayed until the CD4+/CD8+ (double positive, DP) stage of thymocyte development. Expression of a 
TCR by DN thymocytes results in signals that promote commitment to the 
T cell lineage (13). In contrast, expression of a TCRß chain in conjunction with the pre-TCR
protein forms the pre-TCR, which generates signals resulting in commitment to the
ß T cell lineage and transition to the DP stage of thymocyte development (48).
As assembly of TCRß,
and
chain genes occurs in DN thymocytes that are not yet committed to the
ß or 
T cell lineage, it follows that
ß T cells can have completely assembled TCR
and/or
chain genes (1, 911). If productive and expressed, these rearrangements could lead to the expression of aberrant ß
or 
heterodimers, or possibly a 
TCR, in
ß T cells (12, 13). However, TCR
and
chain genes are not expressed in
ß T cells (10). TCR
chain gene expression may be prevented in
ß T cells through the activity of a cis-acting silencer element (14, 15).
The genes encoding the TCR
and
chains lie in a single locus, the TCR
/
locus (16). In the mouse,
100 V
/V
gene segments lie in the 5' region of the locus, followed by the D
and J
gene segments, the TCR
constant region gene (C
), 61 J
gene segments and the TCR
constant region gene (C
) (Fig. 1A). Thus, the TCR
chain gene is embedded between the V
and J
gene segments. Two transcriptional enhancers, the TCR
(E
) and TCR
(E
) enhancers, have been defined in the locus (1720). E
lies in the intron between J
2 and C
and is active in DN thymocytes, promoting germline transcription and rearrangement of TCR
, but not TCR
, chain genes (21). E
, which lies just downstream of the C
gene, is not active in DN thymocytes (22, 23). Rather, E
becomes active in DP thymocytes, promoting germline transcription, V
to J
rearrangement and expression of TCR
chain genes in cells committed to the
ß T cell lineage (22, 24). This temporal activation of E
is responsible for delaying TCR
gene assembly and expression until the DP stage of thymocyte development (22, 23).
All V
and J
gene segments lie in the same transcriptional orientation and thus rearrange by deletion, resulting in excision of the intervening sequences, including the TCR
genes, from the chromosome (Fig. 1A) (25). Although these extrachromosomal circles contain TCR
chain genes and persist in naive
ß T cells, expression of these TCR
chain genes is not observed (9, 10, 26, 27). It is possible that the genetic program of
ß T cell lineage commitment is non-permissive for TCR
gene expression, as appears to be the case with TCR
chain gene expression. Alternatively, silencing TCR
gene expression in
ß T cells may rely on excision of these genes from the chromosome during V
to J
rearrangement.
Here, a CreloxP approach has been used to generate a modified version of the TCR
/
locus (TCR
/
I) in which initial V
to J
rearrangements occur by inversion. To this end, the Cre recombinase was used to invert a 75-kb loxP-flanked region of the TCR
/
locus. This region encompasses all the J
gene segments, C
and E
such that the J
gene segments on the TCR
/
I allele lie in the opposite transcriptional orientation as the V
gene segments. Accordingly, initial V
to J
rearrangements on the TCR
/
I allele occur by inversion, resulting in retention of TCR
genes within the chromosome in mature
ß T cells. T cell development proceeds in an unperturbed fashion in TCR
/
I/I mice despite the chromosomal retention and expression of fully assembled TCR
chain genes in TCR
/
I/I
ß T cells. These findings are discussed in the context of their importance in
ß T cell development and lineage commitment.
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Methods
|
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Generation of targeting constructs
The 5' homology arm of the pTEA5'loxP targeting vector is a DNA fragment extending 3.8 kb 5' of the BglII upstream of the T early alpha promoter (TEAp) (see Supplementary Figure 1, available at International Immunology Online). The 3' homology arm is a 7-kb BglIINsiI fragment (see Supplementary Figure 1, available at International Immunology Online). The 5' and 3' homology arms were cloned into pLNTK to generate the pTEA5'loxP targeting vector (Supplementary Figure 1, available at International Immunology Online) (24). The 5' homology arm of the pE
3'loxP targeting vector is a 5.5-kb SphIBamHI fragment and the 3' homology arm is a 3-kb BamHI fragment (see Supplementary Figure 2, available at International Immunology Online). These 5' and 3' homology arms were introduced into the pLNTK vector to generate the pE
3'loxP targeting vector (Supplementary Figure 2, available at International Immunology Online).
Embryonic stem cells
Cell culture and gene targeting of embryonic stem cells were carried out as previously described (24).
Southern and northern blot analysis
Southern and northern blot analysis of purified genomic DNA and RNA, respectively, was carried out as previously described (24). Previously described probes are as follows: probe 4 (P4) (9), T early alpha (TEA) (28), C
(29), C
(9), C
1 (10), 5'-1 (30), 3' (30), E
KO5' (24), E
KO3' (24) and glyceraldehyde-3-phosphate dehydrogenase. The recombinase activating gene-2 (RAG-2) probe is a 0.9-kb PstIEcoRV genomic DNA fragment. The PI probe is a 0.5-kb fragment extending 3' of the HindIII site immediately downstream of E
. Quantitation was carried out using a Molecular Dynamics phosphoimager and Imagequant software.
Flow cytometric analyses and cell sorting
Flow cytometric analyses of thymocytes, splenocytes and lymph node cells were carried out as previously described (24). The mAbs used were FITC-conjugated anti-CD8, anti-TCR
and anti-CD3
and PE-conjugated anti-CD4, anti-TCRß, anti-V
2 and anti-Thy1.2 which were all obtained from Pharmingen. Flow cytometric cell sorting was carried out using a FACSvantage.
Cell stimulation and hybridoma generation
ß T cells were stimulated either in media containing 5 µg ml1 ConA and 50 U ml1 IL-2 for 2 days or on plates coated with 10 µg ml1 anti-TCRß antibody (H57-597) in PBS and in media containing 50 U ml1 IL-2 for 6 days (31). To generate
ß T cell hybridomas, activated T cells were fused to the BW-1100.129.237 thymoma and selection was carried out as previously described (24). T cell hybridomas that expressed an
ß TCR, as determined by flow cytometric analyses, were analyzed for TCR
chain gene retention and rearrangement.
PCR and sequence analysis
VDJ
1 rearrangements were amplified using a primer 3' of the J
1 gene segment (J
1) with the V
-specific primers DV104S1, DV105S1, ADV7S and ADV17S2, that would amplify V
4, V
5, V
6/V
7 and V
9 rearrangements to J
1, respectively, on 500 ng of genomic DNA obtained either from sorted resting
ß T cells or from TCR
/
I/+ hybridomas as previously described. PCR products were cloned into the pGEM-Teasy vector and sequenced using the T7 primer as previously described (32).
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Results
|
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Generation of the TCR
/
I allele
A modified TCR
/
locus (TCR
/
I) was generated in which initial V
to J
rearrangements occur by inversion, resulting in retention of the TCR
chain genes within the chromosome in mature
ß T cells (Fig. 1). To this end, the pTEA5'loxP vector was used to target a loxP-flanked neomycin resistance gene 5' of the TEAp on a single TCR
/
allele in an embryonic stem (ES) cell line, generating the TEA5'loxP-Neo-loxP allele (Supplementary Figure 1, available at International Immunology Online). The neomycin resistance gene was deleted through transient expression of the Cre recombinase, generating the TEA5'loxP allele, which has a single loxP site 5' of the TEAp (Supplementary Figure 1, available at International Immunology Online). The pE
3'loxP vector was used to target the loxP-flanked neomycin resistance gene downstream of E
on the TEA5'loxP allele, generating the TEA5'loxP-E
3'loxP-Neo-loxP allele (Supplementary Figure 2, available at International Immunology Online). This targeting placed the loxP sites flanking the neomycin resistance gene in the opposite orientation as the loxP site 5' of the TEAp (Supplementary Figure 2, available at International Immunology Online). Thus, expression of the Cre recombinase in this cell resulted in deletion of the loxP-flanked neomycin resistance gene, generating the TEA5'loxP-E
3'loxP allele, followed by inversion of the 75-kb region between the 5' TEA and 3' E
loxP sites, generating the TCR
/
I allele (Fig. 1A; Supplementary Figure 1, available at International Immunology Online). Fidelity of gene targetings and inversion was assessed by Southern blotting, PCR and sequence analyses (Fig. 1A; Supplementary Figures 1 and 2, available at International Immunology Online, and data not shown).
The 75-kb inverted region on the TCR
/
I allele includes the TEAp, all 61 J
gene segments, the C
gene and E
. Thus, the J
gene segments on the TCR
/
I allele are in the opposite transcriptional orientation with respect to the V
gene segments (Fig. 1). As a result, initial V
to J
rearrangements on this allele will occur by inversion (Fig. 1B). Importantly, subsequent V
to J
rearrangements, which could occur by deletion or inversion, will not lead to deletion of TCR
genes from the chromosome (Fig. 1B). Chimeric mice were generated using ES cells harboring the TCR
/
I allele and germline transmission of the TCR
/
I allele was achieved.
Temporal activation of germline TCR
and
transcripts on the TCR
/
I allele
Germline transcription and accessibility of TCR
chain genes is normally activated in DN thymocytes, whereas activation of TCR
chain gene germline transcription and accessibility is delayed until the DP stage of thymocyte development. To determine whether this temporal activation of germline transcription is intact on the TCR
/
I allele, mice homozygous for the TCR
/
I allele (TCR
/
I/I) on a RAG-2/ background without (TCR
/
I/I : RAG-2/) or with (TCR
/
I/I : RAG-2/ : DO11ß) the DO11 TCRß transgene were generated. Thymocytes from TCR
I/I : RAG-2/ mice are blocked at the DN stage of development with the TCR genes in the germline configuration due to the absence of the RAG-2 protein (33). The presence of the DO11 TCRß transgene in the TCR
/
I/I : RAG-2/ : DO11ß mice causes thymocyte developmental progression to the DP stage (8). However, the TCR genes remain in the germline configuration in these DP thymocytes due to the deficiency of RAG-2.
The level of germline TCR
transcripts from the TCR
/
I and TCR
+ alleles in DN thymocytes was similar, as evidenced by northern blot analysis of RNA isolated from TCR
/
I/I : RAG-2/ and TCR
+/+ : RAG-2/ thymocytes, respectively (Fig. 2A). Importantly, germline TCR
gene transcripts were not detected in DN thymocytes from either the TCR
/
I or the TCR
+ alleles (Fig. 2B). This was evidenced by the lack of hybridization of RNA from these cells to either a C
probe or a probe to the TEA exon, which lies immediately downstream of the TEAp (Figs 1A and 2B, data not shown) (28, 34). However, activation of germline TCR
gene transcripts (TEA and C
) on the TCR
/
I allele was observed in DP thymocytes from TCR
/
I/I : RAG-2/ : DO11ß mice (Fig. 2B, data not shown). Furthermore, the level of these transcripts was similar to that observed from the TCR
/
+ allele in TCR
/
+/+ : RAG-2/ : DO11ß DP thymocytes (Fig. 2B). Together, these findings demonstrate that the 75-kb inversion on the TCR
/
I allele has not disrupted the temporal activation of germline TCR
and TCR
chain gene transcription during thymocyte development.
T cell development in TCR
/
I/I mice
TCR
/
I/I, TCR
/
I/+ and TCR
/
+/+ mice have similar numbers of thymocytes with comparable fractions of DN, DP and CD4+ or CD8+ (single positive) cells (Fig. 3A, Table 1). Analyses of spleen and lymph nodes in TCR
/
I/I, TCR
/
I/+ and TCR
/
+/+ mice revealed similar numbers of mature CD4+ and CD8+
ß T cells (Table 1, data not shown). Flow cytometric analyses revealed normal levels of TCR expression by TCR
/
I/I
ß T cells, as evidenced by cell surface TCRß and V
2 expression (Fig. 3B). Furthermore, purified TCR
/
I/I and TCR
/
+/+
ß T cells have similar levels of mature TCR
transcripts (Fig. 3C). TCR
/
+/+ and TCR
/
I/I mice have similar numbers of thymic and splenic 
T cells (Table 1, data not shown). Flow cytometric analyses revealed that TCR
expression by TCR
I/I 
T cells was similar to that of wild type 
T cells (Fig. 3B). Together, these findings demonstrate that the assembly and expression of TCR
and TCR
chain genes on the TCR
/
I allele are intact, permitting the efficient generation of
ß and 
T cells in TCR
/
I/I mice.
TCR
chain genes are diversely rearranged and retained in the chromosome in TCR
/
I/I
ß T cells
TCR
gene rearrangements were assayed in purified peripheral TCR
/
+/+ and TCR
/
I/I
ß T cells and in clonal TCR
I/+
ß T cell hybridomas (Fig. 4). Genomic DNA isolated from mature TCR
/
I/I
ß T cells was digested with BglII and subjected to Southern blot analysis using P4 to detect rearrangements to the J
1 gene segment (Figs 1A and 4A). The TCR
genes are extensively and diversely rearranged in TCR
/
I/I
ß T cells, as evidenced by loss of the germline size and generation of multiple non-germline size P4-hybridizing bands. TCR
/
+/+
ß T cells exhibited a similar pattern of diverse TCR
chain gene rearrangements (Fig. 4A). Analysis of TCR
chain gene rearrangements in TCR
I/+
ß T cell hybridomas revealed that each had a single P4-hybridizing band, demonstrating that a single TCR
chain gene was retained in the chromosome in each of these cells (Fig. 4B). Furthermore, these TCR
chain genes were heterogeneously rearranged as evidenced by the different size P4-hybridizing bands (Fig. 4B).
To determine whether the rearranged TCR
chain genes are retained in the chromosome of TCR
/
I/I
ß T cells, Southern blot analyses were carried out using the C
probe on StuI-digested genomic DNA isolated from resting
ß T cells or
ß T cells that had undergone proliferative expansion (Figs 1A and 4C). TCR
genes on extrachromosomal circles should be lost upon cellular expansion if they are incapable of replicating. As compared with genomic DNA isolated from kidney, the C
-hybridizing StuI fragment was 40% retained in genomic DNA isolated from resting TCR
/
+/+
ß T cells (Fig. 4C). However, there was essentially no detectable C
-hybridizing band in genomic DNA isolated from TCR
/
+/+
ß T cells that had undergone proliferative expansion (Fig. 4C). In striking contrast, resting and expanded TCR
/
I/I
ß T cells both exhibit essentially 100% retention of the C
-hybridizing band (Fig. 4C). Together, these data demonstrate that essentially all the TCR
genes in TCR
/
+/+
ß T cells exist on extrachromosomal circles, whereas those in TCR
/
I/I
ß T cells are retained in the chromosome.
TCR
chain genes are productive and expressed in TCR
/
I/I
ß T cells
Many of the TCR
gene rearrangements in TCR
/
I/I
ß T cells were complete as evidenced by Southern blot and PCR analyses (data not shown). To determine the fraction that were in-frame, complete VDJ
rearrangements from resting TCR
/
I/I and TCR
/
+/+
ß T cells were PCR amplified, cloned and sequenced. These analyses revealed that a similar fraction of complete TCR
gene rearrangements were in-frame in TCR
/
I/I (28%) and TCR
/
+/+ (32%)
ß T cells (Table 2). Together, these analyses reveal that TCR
genes in TCR
/
I/I
ß T cells are diversely rearranged. Many of these rearrangements are complete and productive as is the case with TCR
gene rearrangements in TCR
/
+/+
ß T cells.
Northern blot analysis was performed on RNA isolated from resting TCR
/
I/I and TCR
/
+/+
ß T cells. Both TCR
/
I/I and TCR
/
+/+
ß T cells have silenced TCR
chain gene expression as evidenced by the lack of C
1-hybridizing transcripts (Fig. 5). In contrast, there are robust levels of TCR
transcripts in TCR
/
I/I, but not TCR
/
+/+,
ß T cells (Fig. 5). Remarkably, the level of TCR
transcripts in TCR
/
I/I
ß T cells is similar to that observed in 
T cells (Fig. 5). Thus, TCR
chain genes are expressed in
ß T cells when retained within the chromosomal context (TCR
/
I) but not when they are placed on extrachromosomal circles (TCR
/
+).
 |
Discussion
|
---|
We have generated and analyzed mice with a modified version of the TCR
/
locus (TCR
/
I) in which initial V
to J
rearrangements occur by inversion, resulting in the intervening sequence, including the TCR
genes, being retained within the context of the chromosome. The 75-kb inversion on the TCR
/
I allele includes the TEAp, the J
gene segments, C
and E
(Fig. 1). Importantly, the function of cis-acting elements appears intact on the TCR
/
I allele. Germline TCR
transcripts from the TCR
/
I allele are observed in DN thymocytes, whereas activation of TCR
germline transcripts is delayed until the DP stage, as is observed for the TCR
/
+ allele (Fig. 2). Furthermore, complete TCR
and TCR
chains encoded by the TCR
/
I allele are expressed at normal levels in mature
ß and 
T cells, respectively (Fig. 3). However, unlike TCR
/
+/+
ß T cells, TCR
chain genes are robustly expressed in TCR
/
I/I
ß T cells (Fig. 5).
It has been suggested that excision of TCR
chain genes from the chromosome may be important for
ß T cell development and lineage commitment (25). This would occur through initial rearrangements of V
gene segments, or the delta Rec elements, to J
gene segments (25, 3537). Our findings demonstrate that excision of TCR
chain genes from the chromosome per se is not required for
ß T cell development and lineage commitment. In this regard, development of
ß T cells is generally unperturbed in TCR
/
I/I mice despite the fact that all
ß T cells in these mice maintain both TCR
chain genes in the chromosome (Figs 3 and 4). Furthermore, many of these TCR
chain genes are completely, and productively, rearranged (Table 2).
We find that the frequency of productive TCR
rearrangements in TCR
/
I/I
ß T cells is similar to that observed in TCR
/
+/+
ß T cells even though the TCR
chain genes in the latter exist on extrachromosomal circles and are not expressed (Figs 4 and 5, Table 2). Although it is possible that a small fraction of TCR
chain gene rearrangements are selected against in developing TCR
/
I/I
ß T cells, these findings demonstrate that there is no general selection bias against the expression of productive TCR
chain genes in these cells. It is possible that additional mechanisms may prevent TCR
chain expression in
ß T cells that express TCR
chain genes. For example unpaired TCR
chains may be rapidly degraded, as is observed for unpaired TCR
chains (3840). In this regard, flow cytometric analyses using two pan-TCR
chain mAbs and an anti-V
4 mAb failed to reveal any cell surface or intracellular TCR
chains in TCR
/
I/I
ß T cells (data not shown). This could be due to the inability of these antibodies to recognize TCR
chains outside the context of a 
heterodimer or due to the instability of TCR
chains in these cells.
Our findings demonstrate that the genetic program of developing
ß T cells is permissive for the efficient assembly and expression of a diverse array of complete TCR
chain genes. What then is the mechanistic basis for silencing of TCR
chain gene expression in
ß T cells? It is possible that TCR
chain gene expression in
ß T cells is normally extinguished by a cis-acting silencer element whose function has somehow been disrupted on the TCR
/
I allele. However, robust levels of germline TCR
transcripts are found in DP thymocytes from TCR
/
+/+ : RAG-2/ : DO11ß mice, where the TCR
gene remains in the chromosome (41). Furthermore, completely assembled TCR
chain genes can be expressed in
ß TCR transgenic T cells that have not undergone V
to J
rearrangements on both TCR
alleles (10). Together with our findings these data suggest that TCR
gene expression in
ß T cells is not silenced through the activity of a cis-acting silencer element.
Silencing of TCR
gene expression could be due to the loss of TCR
genes on extrachromosomal circles during cell division. However, these extrachromosomal circles are generated through V
to J
rearrangement in DP thymocytes, which do not divide until they become mature T cells and are activated by antigenic stimulation. Thus, extrachromosomal circles containing complete TCR
chain genes can, and do, persist at significant levels in naive
ß T cells (Fig. 4) (9, 10, 26, 27). Although simply placing TCR
chain genes on extrachromosomal circles may prohibit expression, robust gene expression occurs on plasmid-based circular extrachromosomal substrates that contain appropriate cis-acting elements. In this regard, excision circles generated by V
to J
rearrangement contain entire TCR
chain genes with their promoters and the TCR
enhancer. However, optimal TCR
chain gene expression in 
T cells requires E
and not E
(21, 24). Furthermore, optimal germline TCR
chain gene transcription in DP thymocytes also relies on E
(41). V
to J
rearrangement on the wild type TCR
/
allele places E
, which remains in the chromosome, in trans with TCR
promoters on excised extrachromosomal circles. In contrast, E
and TCR
promoters remain in cis after V
to J
rearrangement on the TCR
/
I allele. Thus, silencing of TCR
gene expression in
ß T cells likely occurs because requisite cis-acting elements, such as E
, are placed in trans with TCR
promoters on extrachromosomal circles.
 |
Supplementary data
|
---|
Supplementary data are available at International Immunology Online.
 |
Acknowledgements
|
---|
B.P.S. is a recipient of an Investigator Award in General Immunology and Cancer Immunology from the Cancer Research Institute. Mice were produced by a transgenic mouse core facility supported by the Rheumatic Diseases Core Center at Washington University (NIH P30-AR48335).
 |
Abbreviations
|
---|
DN | double negative |
DP | double positive |
ES | embryonic stem |
P1 | probe 1 |
RAG | recombinase activating gene |
TEA | T early alpha |
TEAp | T early alpha promoter |
 |
Notes
|
---|
Transmitting editor: D. Littman
Received 8 September 2004,
accepted 1 December 2004.
 |
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