Review

Immunology and Cell Biology (2008) 86, 166–174; doi:10.1038/sj.icb.7100120; published online 23 October 2007

Expression of T-cell receptor genes during early T-cell development

Janice L Abbey1 and Helen C O'Neill1

1School of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT, Australia

Correspondence: Professor HC O'Neill, School of Biochemistry and Molecular Biology, Building #41, Linnaeus Way, Australian National University, Canberra, ACT 0200, Australia. E-mail: Helen.ONeill@anu.edu.au

Received 22 December 2006; Revised 21 August 2007; Accepted 2 September 2007; Published online 23 October 2007.

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Abstract

Lymphoid cell development is an ordered process that begins in the embryo in specific sites and progresses through multiple differentiative steps to production of T- and B-cells. Lymphoid cell production is marked by the rearrangement process, which gives rise to mature cells expressing antigen-specific T-cell receptors (TCR) and immunoglobulins (Ig). While most transcripts arising from TCR or Ig loci reflect fully rearranged genes, germline transcripts have been identified, but these have always been thought to have no specific purpose. Germline transcription from either unrearranged TCR or unrearranged Ig loci was commonly associated with an open chromatin configuration during VDJ recombination. Since only early T and B cells undergo rearrangement, the association of germline transcription with the rearrangement process has served as an appropriate explanation for expression of these transcripts in early T- and B-cell progenitors. However, germline TCR-Vβ8.2 transcripts have now been identified in cells from RAG/ mice, in the absence of the VDJ rearrangement event and recombinase activity. Recent data now suggest that germline TCR-Vβ transcription is a developmentally regulated lymphoid cell phenomenon. Germline transcripts could also encode a protein that plays a functional role during lymphoid cell development. In the least, germline transcripts serve as markers of early lymphoid progenitors.

Keywords:

T cells, T-cell receptor, gene rearrangement, germline transcription

Lymphopoiesis is a vital step in development that equips a repertoire of immune cells to fight and combat infection. The importance of these cells correlates with the level of complexity required to develop the individual types. All immune cells derive from hematopoietic stem cells (HSC), which possess long-term self-renewal capability. HSC progress through multiple developmental pathways and diverge into several lineages. Any given HSC, provided with the appropriate niche and stimulus, can develop into a T- or B-cell, monocyte, neutrophil, eosinophil, basophil, platelet or erythrocyte. This has been demonstrated in numerous reconstitution experiments where irradiated murine hosts are transplanted with HSC.1, 2, 3, 4, 5

The progression of lymphopoiesis is quite distinct in the embryo as opposed to the adult mouse. In the developing embryo, erthyropoiesis predominates in the yolk sac from embryonic days 7 to 12 (Figure 1).6, 7, 8, 9 The fetal liver has been characterized as the major developmental site for hematopoiesis arising from HSC. However, transplantation experiments have also identified multipotent HSC present in the yolk sac.9, 10 HSC are also present in fetal liver at approximately embryonic day 11, and this site predominates as the major developmental site throughout embryogenesis until the bone marrow takes over this role at birth.6, 9, 11 Development and proliferation of HSC in fetal liver continues until embryonic day 15 to 16 when fetal liver activity begins to decline. While HSC appear in the spleen and bone marrow, the bone marrow remains the major site for HSC development in the newborn and adult.9 During the transition of cells from yolk sac to fetal liver, some cells also seed thymus around embryonic days 10–11. Thymus then continues as a major site for T-cell development in the newborn and the adult.2, 10

Figure 1.
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The emergence of HSC in the murine embryo during gestation. HSC are first identified in the yolk sac of the developing embryo. However, by day 11 of embryogenesis, they can be detected in fetal liver. This site predominates as the major site of HSC until just prior to birth (~18 days), when the bone marrow is seeded (~15 days) and takes on this role in the adult. Adapted from Christensen et al1 and Ikuta et al.10

Full figure and legend (63K)

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T-cell development in the thymus

The definitive stages in T-cell development occur in the thymus. In adult mice, bone marrow-derived T-cell precursors enter this organ via blood and undertake strict progression through the multiple thymic environments.12, 13 The progression of cells through three major developmental stages can be traced by expression of the CD4 and CD8 cell surface markers (Figure 2). During development, there is progressive development of double negative (DN) CD4CD8 cells, double positive (DP) CD4+CD8+ cells and single positive (SP) CD4+ or CD8+ cells. The DN stage can be segregated into further developmental stages based on expression of the cell surface markers CD25 and CD4414, 15 (Figure 2). The level of expression of these cell-surface markers fluctuates as the cells move through various niches in the thymic architecture.13, 16 Furthermore, the phenotype of the bone marrow-derived cell that first seeds the thymus is controversial.17

Figure 2.
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The major events in T-cell development in thymus. Cells enter the thymus from the blood as CD4CD8 (double negative; DN) thymocytes. Cells classified as DN thymocytes reflect a heterogeneous population with respect to the expression of CD25 and CD44. Subsets of cells reflect stages in development referred to as DN1 to DN4, where CD44 marker expression is elevated in the DN1 and DN2 stages, but is downregulated in DN3 and DN4 stages. CD25 marker expression occurs within the DN2 and DN3 stage, but is also downregulated in the DN4 stage of thymopoiesis. The expression of TCRβ and pTα molecules on the cell surface is detected in the later stages, DN3 and DN4, prior to progression to the CD4+CD8+ (double positive:DP) stage. DP cells undergo a selection process before progressing to the DP stage of development, where cells express either CD4 or CD8, but not both. Adapted from Sebzda et al15 and von Boehmer.22

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Some researchers have queried the isolation techniques used to purify the DN fraction of thymus, since accessory cells like dendritic cells, fibroblasts and epithelial cells exist in this site, which are also ‘double negative’ for CD4 and CD8 but are not T-cell precursors. For this reason, the additional cell surface marker c-kit (CD117) has been used in the characterization of DN1 cells.16 Analysis of c-Kit as well as CD24 expression among these cells has shown that DN1 thymocytes are not as homogeneous as originally thought. In fact, CD4CD8CD25CD44+ DN1 cells have now been shown to comprise five sub-populations, referred to as DN1a–e.18 Of these multiple populations, only DN1a (CD24-) and DN1b (CD24lo) exhibit homologous characteristics of T-cell precursors including high proliferative activity, similar transcriptional profiles and strong ability to home to the thymus.18 DN1a cells are precursors to the DN1b population. However, both populations have potential to give rise to cells expressing the NK1.1 marker. In contrast, the DN1c (CD24+) and DN1d (c-Kit-CD24+) populations display ability to produce CD19+ B cells.18 This emphasizes the maintenance of pluripotency by early lymphoid progenitors.

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Rearrangement of TCR genes

The rearrangement of TCR genes leading to T-cell receptor (TCR) expression on the cell surface is essential for progression during T-cell development. The TCR comprises an alpha (α) and a beta (β) chain. To produce a functional αβ TCR, multiple exons dispersed along the genomic DNA must be joined and transcribed. This process occurs independently for each chain, with genomic DNA rearrangement or recombination commencing first during ontogeny for TCR-β chain genes located on chromosome 6.19, 20 Each cell contains two copies of the TCR-β locus, one on each allele. However, TCR-β chain gene rearrangement is restricted, ensuring that only one fully rearranged TCRβ chain is produced. The mechanism by which this occurs is referred to as allelic exclusion.

The TCRβ chain comprises four regions encoded by individual gene segments in the TCR-β locus: variable (V), diversity (D), joining (J) and constant (C) region exons. There are approximately 20 segments, two segments, two segments (each comprised of six independent exons) and two regions19, 20, 21 (Figure 3). To produce a functional TCRβ chain, irreversible TCR-β VDJ recombination or rearrangement must occur in the genomic DNA on one chromosome to bring a V, D and J exon together in one open-reading frame (Figure 4). This occurs during the DN2 and DN3 stages of T-cell development and is mediated by enzymes encoded by the recombination activating genes-1 and -2 (RAG-1 and RAG-2, respectively).22 RAG-1 and RAG-2 are essential and specific to the rearrangement of antigen-specific receptors in both B and T cells, and it has been suggested that these lymphoid cells share common RAG enzymes.23 Mice deficient in either RAG-1 or RAG-2 lack mature B and T lymphocytes and cells are arrested in development.24, 25

Figure 3.
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Schematic representation of the mouse TCR-β locus. The TCR-β locus is comprised of approximately 20 functional V genes, each containing a leader (L) region. Downstream of the region, two consecutive blocks of DJC regions are located, each consisting of one , six and one region. A promoter (PDβ1) is situated upstream from Dβ1Jβ1Cβ block and an enhancer is located downstream of Cβ2. Adapted from Jackson and Krangel (2006).29

Full figure and legend (43K)

Figure 4.
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Comparison of VDJ recombination and germline transcription of TCR-Vβ genes. At rearrangement, the mouse TCR-β locus undergoes irreversible change in genomic DNA to give a VDJ rearranged locus prior to transcription of the TCR-β chain. In contrast, transcription has been identified which occurs directly from individual genes prior to the VDJ recombination event. This is referred to as germline transcription from unrearranged or germline genes. Germline transcription commences from the start of the leader (L) region, continues through the exon and is often terminated at a stop codon in the 3′ untranslated region. Termination varies with the sequence of the TCR-Vβ gene, since internal stop codons may exist in either the intron or exon. The primary mRNA is spliced to removed the intron between the L and exon.

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RAG-1 and RAG-2 recognize specific recombination signal sequences (RSS) comprising a heptamer–spacer-nonamer consensus sequence in the DNA. Spacer sequences are either 12 or 23bp in length, and only an RSS with a 12bp spacer can join with an RSS with a 23bp. This is known as the 12/23 rule, which regulates the correct recombination of exons in the TCRβ locus. RSS are located before and after coding sequences of D and J regions and at the 3′ end of V region coding exons. For example, V regions contain a 23bp spacer on the 3′ end while D regions contain a 12bp spacer on the 5′ end and a 23bp spacer on the 3′ end. This enables D regions to recombine with V regions following DJ rearrangement and to form the VDJ complex.21, 22, 26 Hairpin loops are cleaved by endonucleases, and random nucleotide addition by terminal deoxynucleotidyl transferase (TdT), which is used to fill the gap between the D and J region, and between the DJ and V region.21, 27

Transcription of a fully rearranged TCR-β locus occurs from the beginning of the leader region of the V gene, through to the end of the C region. Transcript processing through splicing removes the intervening intron producing a mature TCRβ transcript (Figure 4). The mature transcript is translated to produce a functional TCRβ polypeptide chain. If errors occur during the recombination process, TCR-β gene rearrangement can continue on the same chromosome, until all available exons have been attempted. In the case where rearrangement is unsuccessful, TCR-β locus rearrangement will begin on the second chromosome.27, 28

Unusual splicing of TCR-Vβ genes has been documented for the Vβ8.2 gene. Rearrangement of this gene with proximal and distal D, J and C regions occurs by the mechanism described. However, transcription of this gene predominantly initiates from the leader of the Vβ5.1 gene, located directly upstream of Vβ8.2.20 This phenomenon is thought to reflect promoter strength of the leader5.1 region in comparison to leader 8.2. Unusual splicing of TCR-Vβ genes has only been identified for TCR-Vβ8.2, and rearranged products from this gene arise predominantly from the leader 5.1 promoter. Upon production of a functional TCRβ polypeptide chain, a surrogate pre-TCRα (pTα) complex is expressed on the cell surface. Signalling through the pTα/TCRβ complex maintains thymocyte survival and promotes proliferation and progression to the DP stage of development.29

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Allelic exclusion

Allelic exclusion is a regulatory mechanism that controls expression of a single TCRβ chain within a cell. While the potential exists for two rearranged TCRβ chains to be expressed simultaneously, in practice this does not readily occur. It is well documented that VDJ recombination of the TCR-β locus is more tightly regulated at particular stages of development. While to recombination occurs on both chromosomes, to DJβ recombination proceeds on only one.30 This type of evidence defines allelic exclusion and specific regulation of gene rearrangement. One model is that if the first rearrangement event of to DJβ is unsuccessful and out-of-frame, further genes on the same chromosome can be targets for rearrangement. Contrary evidence suggests that after a first attempt is made on one chromosome, the next is made on the second chromosome if it has undergone productive Dβ-Jβ rearrangement.30 This poses questions about the regulation of TCR-β locus accessibility and enforcement of allelic exclusion within the TCR-β locus once both alleles are made accessible. In addition, as cells progress to the DP stage of development, a second wave of RAG expression is induced. One question then is how does the accessible TCRβ locus avoid further rearrangement in the presence of RAG enzymes.21, 22, 31 The mechanism that makes genes inaccessible is not clear. These issues surrounding allelic exclusion remain a continuing topic of investigation. The current model is multifaceted and involves feedback inhibition mechanisms by signalling through the pTα/TCRβ complex,32, 33 together with accessibility regulation by epigenetic tools, including histone acetylation, DNA methylation, nuclease sensitivity and nuclear positioning.

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Feedback inhibition

The role of feedback inhibition in allelic exclusion has been demonstrated indirectly in experiments involving pTα/ mice. A study by Mancini et al34 showed an 11-fold decrease in the percentage of DP cells in the thymus of embryonic day 18 pTα/ mice compared with wild type mice. In addition, the TCR-α locus was open and accessible for rearrangement events with no sign of regulation. It is now known that progression through the β-selection checkpoint was impaired in pTα/ mice due to substantial cell loss by apoptosis.35 The level of regulation of locus accessibility was lower in pTα/ mice compared with wild type mice, thus implicating pTα signalling in the regulation of allelic exclusion prior to the β-selection checkpoint.

Further evidence demonstrating a role for pTα and signalling components in allelic exclusion involves the intracellular signalling molecule LAT (linker for activation of T cells).31 LAT/ mice display a block in thymocyte development between the DN and DP stage. LAT was shown to be important in allelic exclusion by experiments analysing rearrangement of endogenous TCR-β genes in LAT/ mice carrying a rearranged TCR-β transgene. The presence of the TCR-β transgene in LAT/ mice did not alleviate the DN to DP block but led to upregulation of to DJβ rearrangements by comparison with LAT+/ thymocytes carrying the same transgene.31 Analogous feedback inhibition mechanisms have also been associated with Lck, SLP-76,36, 37, 38 Raf and PKCα.31, 39 Feedback inhibition mechanisms that proceed signalling through the pTα/TCRβ/CD3 complex appear to regulate allelic exclusion.

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Epigenetic regulation: histone modification & DNA methylation

Accessibility of DNA both prior to and during recombination has been attributed in part to histone modification. Genomic DNA is packaged into dense chromatin structures with DNA tightly complexed and inaccessible to binding proteins. Approximately 150bp of genomic DNA is wrapped around a nucleosome complex comprising eight subunits: two H2A/B dimers plus one H3/H4 tetramer. Nucleosome structures are further complexed into chromatin.40, 41, 42 Therefore, DNA is tightly bound and inaccessible to RAG enzymes and transcription factors, and must be unravelled before these proteins can gain access. DNA accessibility during VDJ recombination, and access of RAG enzymes to the DNA through the nucleosome complexes, are major determinants of recombination, although other levels of regulation beyond accessibility have also been described.43

Recently, West et al44 demonstrated that RAG-2 interacts directly with components of the nucleosome. Binding of RAG-2 through the C terminus to the nucleosome components H2A, H2B, H3 and H4, was demonstrated by immunoprecipitation and in vitro binding assays. Therefore, RAG-2 not only binds to RAG-1 to form the active complex, but also binds directly to nucleosomes. Analysis of the effect of C-terminal mutations of RAG-2 in recombination of Ig genes, revealed a lower D to J recombination, but very reduced V to DJ recombination.

Specific protein modification of histones can result in DNA release from the nucleosome complex and accessibility to transcription factors.42, 45, 46 This has been demonstrated at the mouse TCR-γ locus and specifically for Vγ2 and Vγ3 genes. These show differential expression during development,47 which is thought to correlate with gene accessibility regulated by the level of histone acetylation. Acetylation of histones results in alteration in the binding interaction between DNA and histones, leading to conformational change and accessibility. When histone deacetylase was inhibited with trichostatin A, the level of DNA accessibility and rearrangement at the TCR-Vγ locus was increased.47 Changes in TCR-β locus accessibility have been directly linked with acetylation of H3 histone and other gene transcription, thought to be indicative of an open chromatin structure.48

Modification of DNA has also been shown to affect accessibility of the DNA. Genomic DNA exists in a methylated state, and evidence has accumulated linking DNA demethylation with DNA/protein interactions and allelic exclusion. For example, Goldmit et al49 demonstrated that the mouse Igκ locus is demethylated in a monoallelic fashion, with increased sensitivity to DNase I treatment and higher affinity for double-stranded breaks. Therefore, the methylation state of the DNA may play a significant role in DNA accessibility both prior to and following rearrangement.50

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Cis-Regulatory elements: enhancers and promoters

Studies investigating phenotypic changes in mice with defective or deleted enhancers and promoters of TCR genes have provided significant insight into the potential role of these elements in T-cell development. The TCR-β locus contains an enhancer approximately 5.9kb upstream from Cβ2 and 2.9kb downstream from Vβ14 (Figure 3). This enhancer regulates Dβ1 to Jβ1 recombination, and to a lesser extent, Dβ2 to Jβ2 recombination. Bouvier et al51 examined the effect of deletion in homozygous / mice and showed that the levels of DP and SP cells in thymus were dramatically reduced, with TCR-β rearrangements undetectable. disruption also has a significant effect on the DN and DP populations and specifically Dβ1 and Jβ1 recombination.52 The same effect was also reported for Dβ2 and Jβ2, with a 25-fold decrease in rearrangement in comparison with wild type. Unusual to rearrangements were also detected when Dβ1 was involved, giving Dβ1–Dβ2–Jβ2 rearrangements. This demonstrates the important role of the enhancer in VDJ recombination and suggests a direct role in the rearrangement of and segments. While Dβ1 and Dβ2 remained in germline configuration in DN cells, a small percentage of DP cells underwent rearrangement. may play a role in regulating VDJ recombination at and regions, and may regulate the accessibility of these gene segments in DN cells. The role of in accessibility of DβJβ regions has been shown previously. However, it was also found that did not affect the accessibility of regions.53 Since the distance of regulatory control for is thought to be within ~25kb on either side of the enhancer,53 this could explain the normal phenotype of in / mice.

The inability of to affect was investigated using a mutant RAG-2/ mouse model lacking all genes except Vβ10, Vβ4, Vβ16 and Vβ2.54 In this model, a large proportion of the region from Vβ10 to Dβ1Jβ1 was deleted. This reduced the distance between Vβ10 and , putting Vβ10 into the effective zone of . When / and +/+ mice were compared, a difference in the level of accessibility for Vβ10 was identified. Accessibility was conferred through H3 histone acetylation, suggesting that modulates its effect through positioning, confirmed by preferential Vβ10 rearrangements in comparison with wild type mice. Unusual rearrangements were also detected in double mutant mice, with V to J joins identified. However, V to D remained the prominent form. This confirms the directive role of in VDJ recombination and the significance of position in TCR-β locus accessibility.

The TCR-β locus contains a well-characterized promoter region involved in the regulation of VDJ recombination. The Dβ1 promoter (PDβ1) is located 400bp upstream of Dβ1 exons in the TCR-β locus.55 This promoter has been widely studied by mutational or deletional analysis and found to significantly affect VDJ recombination and accessibility. Following mutation, events involving Dβ1 and Jβ1 gene segments were detected in the mutant at a level of ~1% in comparison to wild type mice. The effect of PDβ1 is specific to Dβ1 gene rearrangement and accessibility, since Dβ2 and Jβ2 gene rearrangement remained unaffected. This effect also extends to and DJβ joining.56 Additional studies examining a mutant PDβ1 promoter using mini-loci also noted a decreased effect on VDJ recombination in cells lacking this promoter but containing an enhancer. Restoration of VDJ recombination was possible if the PDβ1 or another promoter element was reintroduced.57 This demonstrates how TCR-β promoters rely on enhancers to control VDJ recombination and regulate chromatin accessibility. Disruption to the PDβ1 promoter not only interferes with Dβ1 recombination but also appears to affect accessibility through the inhibition of histone acetylation and DNA demethylation.58 In comparison with wild type mice, PDβ1/ mice had a 50% decrease in the level of acetylation of H3 in the Dβ1Jβ1 region, and the Dβ1 region was hyper-methylated. In contrast, Sikes et al59 suggest that accessibility of DNA for recombination cannot be entirely conferred by histone acetylation. In mutational studies using a TCR-β mini-locus, it was determined that in the presence of both an enhancer and a promoter, H3 and H4 acetylation levels were high. If the enhancer was deleted, H3 and H4 levels were reduced. In contrast, if the promoter was deleted or relocated between D and J, then no effect was observed on the level of H3 and H4 acetylation. While acetylation levels are unaffected, VDJ recombination levels are lowered, suggesting that the promoter does not directly influence accessibility and mechanisms other than acetylation must influence VDJ recombinase access.59

Position of the PDβ1 promoter was also found to be important in regulating VDJ recombination. When the PDβ1 promoter was relocated either upstream of the Dβ1 region (D-P-J configuration) or upstream of the Jβ1 region (D-J-P configuration), relocation back to the former position resulted in transcriptional levels comparable to wild type. By comparison, transcription was completely ablated when the promoter was in the D-J-P configuration.59 The positioning of the PDβ1 promoter in regard to Dβ1 seems to affect the level of accessibility for VDJ recombination. Oestreich et al60 have provided a model for the interaction of and the PDβ1 promoter. The model requires that DNA bends to allow formation of a holocomplex between the enhancer and promoter. Support for this model comes from experiments examining transcription factor binding at promoter and enhancer sequences. Analysis revealed that SP1, TBP, RNA Pol II, CBP and ATF, specifically bind to either enhancer and/or promoter sequences. Similarities between transcription binding factors have led to the hypothesis that holocomplex formation is favoured. However, it is also possible that each region can bind these factors independently of the other. To examine this possibility, PDβ1/ mice that also lacked the SP1 binding site were used. SP1 binding was inhibited at both PDβ1 and , suggesting that may not bind this factor independently of PDβ1 and may interact through a complex formed with PDβ1.60

Additional promoters in the TCRβ locus are associated with each of the individual genes.61 These are involved in regulation of rearrangement but not allelic exclusion. The effect of deletion of a promoter on TCR gene rearrangement and allelic exclusion has been studied for Vβ13.62 While the CD4+ and CD8+ T-cell population in thymus remained unchanged, the number of cells expressing TCR–Vβ13 was reduced. Rearrangements involving Vβ13 were reduced 5- to 10-fold in mutant mice, while rearrangements involving the neighbouring Vβ8.1 and Vβ12 genes remained unaffected. When mutant mice were transfected with a functionally rearranged TCR transgene, further rearrangement of Vβ13 and neighbouring genes was inhibited in mutant mice and allelic exclusion was maintained. The detection of signal ends can be used as a means of determining whether cleavage has occurred with VDJ recombination. The presence of signal ends was measured in wild type and mutant mice, and a threefold reduction in Vβ13 signal ends was observed in the mutant, with a 15- to 20-fold reduction in the mutant carrying a transgene. However, levels of rearrangement in the mutant may result from lower ability to cleave Vβ13 regions. While promoters may regulate accessibility of DNA for cleavage by recombinase complexes, promoters are not involved in allelic exclusion.

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Germline transcription

Germline transcription is the production of mRNA transcripts from unrearranged TCR or Ig loci, which are in ‘germline’ configuration. A comparison of the mechanism of transcription between a germline and rearranged TCR-β locus is shown in Figure 4. The common view is that the production of germline transcripts is tightly coupled to rearrangement events that take place at these loci to produce a functional TCR or BCR. Recent information at the TCR-α locus also indicates that transcription can regulate recombination and prevent activation of downstream promoters.63 By this model, one would expect germline transcripts to be produced just prior to VDJ recombination when the chromatin is open and fully accessible to the transcriptional machinery. Indeed, germline TCR-Vβ transcripts have been identified in mice well before the TCR-Vβ rearrangement event, when TCR rearrangement is blocked64, 65, 66 and in cells expressing a fully rearranged TCR.67 This evidence questions an association between the TCR rearrangement process and the germline transcription event. In addition, germline transcripts are thought to be sterile and non-functional. However, it now appears that transcripts may encode a TCR-Vβ molecule that can be expressed on the cell surface in the absence of a Cβ region.66, 68 This is a novel finding since the Vβ domain confers antigen specificity while the Cβ domain anchors the receptor in the membrane. This raises many questions about the functional role of germline TCR-Vβ transcripts within the cell, whether they are translated and whether they play a functional role in vivo.

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Evidence of germline transcription of Ig and TCR genes

Germline transcription of TCR-Vβ genes has been reported by several laboratories.64, 65, 66, 69, 70 Candeias et al64 examined the ontogeny of germline TCR-Vβ transcription and suggested a developmental link between the movement of T cells through the developing embryo and the expression of germline TCR transcripts. Germline transcription was not detected in the yolk sac of the developing embryo at any stage of the analysis and was first detected at day 12 in fetal liver. At this time, both T and B cell progenitors are present in fetal liver. In addition, germline TCR-Vβ8.2 transcripts were detected in fetal thymus at embryonic day 14, when only immature lymphoid cells are present in the thymus. These results suggest that germline TCR gene transcription is related to the lymphoid lineage rather than to hematopoiesis in general, since expression was not detected in the yolk sac where HSC are located. Germline transcription may well be an event occurring prior to the rearrangement event at the TCR-β locus.

Several laboratories have investigated the link between germline transcription and VDJ recombination. A model proposed by Yancopoulos and Alt71 explained the coupled expression of germline Ig transcription and VDJ recombination. This ‘accessibility model’ remains the favoured explanation for the presence of germline transcription in vivo. The model relies on developmental and tissue-specific expression of germline transcripts occurring prior to VDJ recombination, when the genomic DNA is accessible and ready for VDJ recombination. By this model, germline transcripts are produced only prior to VDJ recombination and during T or B cell development. Support for the accessibility model comes from several laboratories over the past 20 years. Evidence comes from data on transcription of many germline Ig and TCR genes, including Ig-VH,71, 72, 73 Ig-Cμ,73 Ig-Cκ,74, 75, 76 Ig-Vκ,77, 78 Ig-Vλ,77 TCR-Vα,79, 80 TCR-Vγ and TCR-Cγ.81

Some evidence now shows germline TCR-Vβ transcription occurring independently of VDJ recombination. Germline TCR-Vβ transcription can occur in mutant mouse models lacking either of the RAG-1/2 enzymes required for VDJ recombination. These mice contain T and B cell progenitors that have TCR and Ig genes in germline configuration. The absence of either RAG-1 or RAG-2 results in developmental arrest of lymphocytes in these mice. The prevalence of germline TCR-Vβ8.2 in RAG-1/ mice was found to be high in thymus, spleen, liver and bone marrow.69 Similar results were obtained by Candeias et al64 using RAG-2/ mice, but no germline TCR-Vβ transcripts were detected in the liver. In addition, SCID mice, which lack both mature T and B lymphocytes,82 also produce germline transcripts in thymus and bone marrow. More recent studies by Chen et al65 showed that germline transcription in both RAG-2/ and SCID mice involved many genes in the absence of rearrangement. In addition, germline transcription was also detected in MHC/ mice lacking mature T cells. While this study failed to discriminate between germline and rearranged transcripts, it provided data corroborating the independence of germline transcription and VDJ recombination.

Recent findings on germline transcription of Vα genes also dissociate germline transcription and VDJ recombination. Hozumi et al83 showed that germline Jα49 was developmentally expressed but regulated independently of rearranged genes. Rearranged TCR-α transcripts were produced in the absence of germline Jα49 transcripts in TCR-αβ transgenic mice.83 This has also been shown for Ig-VH genes.84 Cell lines derived from RAG/ mice were used to characterize germline Ig-VH transcription, and recombination activity was restored in these lines by transfection of RAG-1 or RAG-2 genes. VH genes expressed as germline transcripts were not those genes subsequently involved in VDJ recombination in cloned cell lines.84 This type of data uncouples the linkage between germline transcription of specific Ig or TCR genes and specific VDJ recombination events. In addition, studies using transgenic mice carrying a TCR-β minilocus or VDJ minichromosomes transfected into a lymphoid cell line, have demonstrated VDJ rearrangement in the absence of germline transcription and germline transcription independent of VDJ recombination.85, 86, 87 Furthermore, the data suggested that germline transcription was not sufficient to confer accessibility to the required level for VDJ recombination. This further supports the independent relationship between VDJ recombination and germline transcription.86, 87

Further data that dispute the relationship between germline transcription and VDJ recombination comes from an analysis of the Igκ locus. This locus is demethylated in preparation for VDJ recombination. Demethylation of Igκ genomic DNA increases chromatin accessibility and promotes VDJ recombination. For the Igκ locus, demethylation has been shown to be monoallelic, such that only one allele is demethylated at any time.49 This is unusual, since germline transcription at the Igκ locus has been shown to be biallelic.88 Monoallelic demethylation of the Igκ locus supports allelic exclusion models for recombination. However, bialleic germline transcription is not consistent with the accessibility model proposed by Yancopoulos and Alt.71 Singh et al88 have also questioned the idea that germline transcription is directive, occurring on the allele that first undergoes rearrangement. Additional mechanisms such as histone acetylation would be needed to regulate accessibility if germline transcription occurred in a biallelic fashion. Histone acetylation associated with production of germline transcripts has been documented previously.47 However, the interdependence of all these factors is not known. Questions abound regarding the relationship between germline transcription and VDJ recombination. However, regardless of this link, it has already been established that germline transcription is expressed during early stages of lymphoid cell development in advance of VDJ recombination.

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The role of distal promoters and enhancers in germline transcription

Germline transcription has predominantly been studied in relation to VDJ recombination. Both recombination and accessibility are tightly coupled to the function of enhancers and promoters. Germline Dβ1Jβ1 transcripts have been identified prior to rearrangement.55, 89 The germline promoter PDβ1 is required for Dβ1Jβ1 germline transcription but not Jβ2 transcription, since homozygous deletion in mice prevents the generation of Dβ1Jβ1 germline transcripts.56, 58 In the case of a deleted PDβ1 promoter, reinstatement of germline transcription was possible by insertion of an alternative promoter into the minilocus, even in the absence of an enhancer.57 In a further study involving mice with a mutation in the promoter of Vβ13 (PVβ13/), rearrangement was specifically inhibited for Vβ13 but not for adjacent Vβ8.1 and Vβ12.62 In contrast, germline transcription of Vβ13 was only partially inhibited, with no effect on germline transcription of Vβ8.1 and Vβ12. Germline transcription appears to occur independently of rearrangement and may involve other promoters. This is also evident in the case of allelic exclusion, where expression of a TCR-transgene in PVβ13/ mice, inhibits further rearrangement of Vβ13, Vβ8.1 and Vβ12, but not the germline transcription of these genes.62

The combined data suggest that germline transcription, accessibility and promoter function are closely related. This question was addressed further by investigations using a TCR minilocus containing the PDβ promoter sequence 5′ of Dβ1 in either forward or reverse orientation.59 Cells carrying the promoter in reverse orientation displayed a reduction in the generation of germline JβCμ transcripts, at levels similar to those lacking the promoter. It was then demonstrated that the level of accessibility of the promoters was equivalent regardless of orientation, while the production of germline transcripts was significantly different. Sikes et al59 suggest that germline transcript levels are not reliable indicators of the level of accessible or inaccessible chromatin structure.

Germline transcription has also been identified in mice lacking the enhancer. In / mice, rearrangement was not detected, although low levels of germline were observed.51 Germline Jβ1 and Jβ2 transcripts have also been identified in thymocytes of RAG/ mice but not RAG// double mutant mice. In contrast, Vβ5.2, Vβ11 and Vβ14 germline transcripts were produced in RAG/ mice as well as in / mice and in double mutant mice.53 Promoters that regulate and activate transcription from rearranged TCR genes are well characterized.61 It was assumed for some time that the proximal promoters used to initiate transcription from rearranged TCR genes also activate and regulate germline transcription. Chen et al65 demonstrated the use of proximal promoters for the induction of germline Vβ4.1, Vβ8.2 and Vβ14.1 transcripts in thymocytes from both SCID and B6 mice. Five clones of Vβ8.2 were identified, one with a much longer 5’ region. This initiation site was estimated to be approximately −380bp from the ATG start site. This suggests that the majority of germline transcripts are produced from proximal promoters. However, the potential also exists for transcription from a site upstream of the standard proximal promoter.

Distal promoters that regulate germline transcription have been identified for Vβ5.1 and Vβ8.1 genes.90 These were located ~400bp upstream of the ATG start site and shown to be enhancer-independent. Activation of transcription from distal promoters is specific, occurring early in embryonic development before expression of rearranged TCR-β genes and specifically in lymphoid tissues. Both germline and rearranged TCR transcripts are produced from these promoters. The possibility of alternative open-reading frames (ORF) was investigated for both Vβ5.1 and Vβ8.1. One ORF was identified for Vβ5.1 and two for Vβ8.1.90 The alternative ORF for Vβ5.1 was generated from the distal promoter but was also in frame with the transcript generated from the proximal promoter. Therefore, if this long transcript was translated, it would potentially encode a protein with an additional 23 amino acids at the N terminus and would be expected to be functionally distinct.90

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Examples of aberrant TCR chain expression

Germline transcription of Ig and TCR genes is prevalent in early lymphoid cell development. One question is whether germline transcripts are translated and functional in T-cell development. Jolly and O’Neill66 reported the binding of antibody to Vβ8.2 by cells of the lymphoid precursor line, C1-V13D. Since antibody to Cβ did not bind, this suggested the expression of an aberrant germline-encoded Vβ8.2 chain. A cell population with the Vβ8.2+ phenotype was later identified in the mesenteric lymph node of DBA/2j mice, representing ~4% of cells.70

Aberrant immunoglobulin receptors have also been demonstrated on the cell surface. Ucker et al91 identified a ~12kDa germline-encoded VH protein in a cloned cytotoxic T lymphocyte (CTL) cell line. Analysis of mRNA revealed that germline VH transcripts were generated from an unrearranged Ig heavy chain V region. While there was no transmembrane region identified in the sequence, a signal sequence specific for secretion was evident. This suggests that individual V genes can be transcribed and that they do have the potential to encode unique and novel proteins. The specific function of this protein was not defined.

There have been several reports of unusual TCR structures expressed by immature T cells. TCRβ chain expression has been detected in the absence of α, γ or δ, chains and TCRβ homodimer expression has been reported.92, 93 However, with the identification of pTα,94 reports of TCRβ chain expression in the absence of a mature TCRα chain were reconsidered, but not all reports of aberrant expression could be explained by the presence of pTα. Balomenos et al95 identified expression of in-frame dual TCRβ chains in C57BL/6 and BALB/c splenic T cells, where increased expression correlated with increasing age of the mice. In addition, Vβ3+Vβ8+ cells showing dual β chain expression were responsive and expansive to stimulation with Staphylococcus enterotoxin B, which specifically activates TCR-Vβ8 receptors.96, 97 Several reports have identified dual Vβ expression in humans98, 99 and dual TCRγ receptor expression.100 The expression of a rearranged Vβ chain on the cell surface of bone marrow and peripheral blood lymphocytes in the absence of pTα or CD3 (other than CD3δ), has also been documented,101 although in a transgenic mouse model.

The expression of dual TCR genes has also been documented for TCR-Vα.102, 103, 104 A population of cells expressing Vα2Vα8 was identified in the mesenteric lymph node of C57BL/10 mice,102 but not among the immature thymocyte population. Dual expression of TCR chains may reflect dysfunctional allelic exclusion.

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Is germline transcription restricted to lymphoid cells?

Numerous studies now confirm the prevalence of germline transcripts from many different genes in cells of the lymphoid lineage. Early studies in this laboratory focused mainly on germline Vβ8.2 transcription by the retrovirally transformed cell line, C1-V13D. This cell line has the phenotype of an early T cell: Thy1CD4CD8TCRαβCD3loMac-1CD45+CD44+NK1.1.105 Upon intra-thymic transfer, C1-V13D underwent further T-cell differentiation expressing rearranged TCRαβ, CD3ε, Thy1, CD4 and CD8. Further studies with defined cell subsets and cell lines have revealed that germline TCR-Vβ8.2 transcripts are prevalent in early T cells, mature T cells, early B cells but not pre-B or mature B cells.64, 69 Germline TCR-Vβ transcripts were not detected in cell lines of the monocyte-macrophage lineage, thymic epithelia, mastocytomas, thymomas or fibroblasts.64, 69 These data suggest that germline TCR-Vβ transcription occurs only in cells of the lymphoid lineage representing an early stage of lymphoid cell development.

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Conclusion

Specific factors and proteins regulate the differentiation and commitment of different hematopoietic cell lineages. Since germline transcription has been defined as a lymphoid-related phenomenon, the possibility exists that the transcription of germline Ig or TCR genes may represent a commitment to the lymphoid lineage, or in the case of protein expression, a marker of lymphoid progenitors. While germline transcription may reflect open chromatin configuration, evidence is accumulating to suggest that germline transcription is not simply a by-product of the rearrangement process as proposed originally by Yancopoulos and Alt.71 While early T cells and early B cells, but not mature B cells, have been shown to express germline TCR-Vβ transcripts, the earliest stage in development, where germline transcripts are expressed, must therefore reflect progenitors with potential for T and B cells. The hypothesis investigated here is that germline transcription, and germline-encoded proteins expressed on the cell surface, reflect a developmental stage in early lymphoid cell development.

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