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The Journal of Immunology, 2005, 175: 6659-6667.
Copyright © 2005 by The American Association of Immunologists

The TCR{alpha} Locus Control Region Specifies Thymic, But Not Peripheral, Patterns of TCR{alpha} Gene Expression1

Faith Harrow and Benjamin D. Ortiz2

Department of Biological Sciences, City University of New York, Hunter College, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The molecular mechanisms ensuring the ordered expression of TCR genes are critical for proper T cell development. The mouse TCR {alpha}-chain gene locus contains a cis-acting locus control region (LCR) that has been shown to direct integration site-independent, lymphoid organ-specific expression of transgenes in vivo. However, the fine cell type specificity and developmental timing of TCR{alpha} LCR activity are both still unknown. To address these questions, we established a transgenic reporter model of TCR{alpha} LCR function that allows for analysis of LCR activity in individual cells by the use of flow cytometry. In this study we report the activation of TCR{alpha} LCR activity at the CD4–CD8–CD25–CD44– stage of thymocyte development that coincides with the onset of endogenous TCR{alpha} gene rearrangement and expression. Surprisingly, TCR{alpha} LCR activity appears to decrease in peripheral T cells where TCR{alpha} mRNA is normally up-regulated. Furthermore, LCR-linked transgene activity is evident in {gamma}{delta} T cells and B cells. These data show that the LCR has all the elements required to reliably reproduce a developmentally correct TCR{alpha}-like expression pattern during thymic development and unexpectedly indicate that separate gene regulatory mechanisms are acting on the TCR{alpha} gene in peripheral T cells to ensure its high level and fine cell type-specific expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Successful rearrangement and ordered expression of the TCR genes are crucial for the development of mature, functional T cells. These processes are therefore strictly regulated during thymocyte development. The TCR{alpha} gene encodes a protein chain that is specifically expressed on the surface of {alpha}{beta} T cells as a heterodimer with the TCR {beta}-chain. Interestingly, the TCR{alpha} gene shares a locus with two other genes, namely, those encoding the TCR {delta}-chain and defender against death-1 (Dad1),3 both of which have very distinct expression patterns (1, 2). Although Dad1, a subunit of an oligosaccharyl transferase enzyme (3), is expressed ubiquitously, TCR{delta} is expressed exclusively in {gamma}{delta} T cells.

Located between the TCR{alpha} gene segments on the 5' end and Dad1 on the 3' end is the TCR{alpha} locus control region (LCR) (4) (see Fig. 1A). An LCR is operationally defined as a cis-acting gene regulatory element that imparts tissue-specific, copy number-dependent expression upon a linked reporter gene independent of the site of genomic integration. The first LCR to be described was that of the human {beta}-globin gene cluster (5). Several other immunologically important genes are now known to be regulated by LCRs (6, 7, 8, 9, 10, 11, 12). LCRs are characterized by clusters of DNase I-hypersensitive sites (HS) that must work collectively to achieve complete LCR activity. The LCR at the TCR{alpha}{delta} locus consists of nine HS, namely, HS1 through HS8 and HS1' (13).



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  		FIGURE 1. A, Scale diagram of the 3' end of the TCR{alpha}/Dad1 locus. The positions of hypersensitive sites of the TCR{alpha} LCR are indicated by vertical arrows. The transcriptional orientations of the TCR{alpha} and Dad 1 genes are indicated by horizontal arrows. {blacksquare}, Exon sequences; C{alpha}, TCR{alpha} constant region exons; E{alpha}, TCR{alpha} enhancer. B, Diagram of the hCD2:1-8 transgene construct. The hCD2 promoter and exons ({square}) are linked 5' of the TCR{alpha} LCR hypersensitive sites. The premature stop codon in exon V is indicated by the vertical arrow. C, Diagram of the {beta}:1-8 construct. The human {beta}-globin reporter gene is linked 5' of the TCR{alpha} LCR hypersensitive sites.

 
Previously, in vivo studies of TCR{alpha} LCR activity made use of transgenic mouse models in which a 4.9-kb BglII genomic fragment of human {beta}-globin was used as a reporter (13, 14, 15, 16). This fragment, when linked to the wild-type LCR, was expressed lymphoid specifically in thymus and spleen. This model only allowed for analysis of {beta}-globin mRNA expression in bulk tissues. In this study we have developed a new transgenic model using a modified version of the human CD2 (hCD2) gene as a reporter. Normal hCD2 functions include mediating T cell adhesion and signal transduction through its cytoplasmic domain (17, 18). The hCD2 reporter gene used in this study has been modified by the insertion of a stop codon into exon V of the gene, resulting in a protein with a truncated cytoplasmic tail, thereby precluding any intracellular signaling by this protein in the T cells of transgenic mice (19). The surface expression of the hCD2 reporter molecule makes possible the analysis of LCR activity in individual cells by flow cytometry. In this study we use this system to address important questions of the fine cell type and developmental stage specificities of TCR{alpha} LCR activity by examination of transgene expression in various hemopoietic lineages and during T cell development in the thymus.

The most immature double-negative (DN; CD4–CD8–) stage of thymocyte development can be further divided into four developmental stages based on surface expression of CD25 and CD44 (20, 21). In this report we show the activation of TCR{alpha} LCR activity at the final DN stage, DN4 (CD4–CD8–CD25–CD44–), coincident with the initiation of endogenous TCR{alpha} rearrangement and expression (20, 22). Moreover, this activity was sustained throughout thymic development. In contrast, the LCR appears insufficient to support several aspects of the normal TCR{alpha} expression pattern in the periphery. Although the endogenous TCR{alpha} gene is up-regulated in peripheral T cells, our studies unexpectedly revealed significantly lower TCR{alpha} LCR activity in mature, resting peripheral T cells compared with that in thymocytes. Furthermore, we report the presence of TCR{alpha} LCR-driven reporter gene expression in {gamma}{delta} T cells and non-T cell hemopoietic lineages, indicating that the LCR is insufficient to restrict linked gene expression to T lineage cells. Our observations demonstrate that the TCR{alpha} LCR contains all the elements necessary to support integration site-independent transgene expression in the thymus with proper, TCR{alpha}-like developmental timing. However, these studies also reveal the requirement of other, as yet unidentified, gene regulatory elements in the wider TCR{alpha}{delta} locus for peripheral T cell up-regulation and further cell type restriction of TCR{alpha} gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Transgenic mice

These transgenic animal studies have been reviewed and approved by the Hunter College institutional animal care and use committee. DNA fragments for microinjection were twice gel purified from low melting agarose (Seaplaque) digested with {beta}-agarase (New England Biolabs). Purified DNA was injected into the male pronucleus of F1 (C57BL/6 x CBA) fertilized mouse eggs that were then injected into pseudopregnant female mice. Potential founders were screened by PCR of DNA derived from ear-punch biopsy. Transgenic founders were outcrossed to C57BL (Taconic Farms) to establish individual lines. Heterozygous transgenic offspring were used for experimental analyses. The relative transgene copy number was determined by PhosphorImager (Molecular Dynamics) analysis of Southern blots. A minimum of three DNA samples purified from tails of individual mice within each line were treated with identical restriction enzymes and hybridized to the same probe on a single Southern blot. Enzymes chosen for restriction analysis and probe preparation allowed for the simultaneous detection of nonoverlapping fragments from the endogenous locus and the transgene. To control for differences in loading, transgene signals were normalized to the endogenous signal within each sample.

Reporter transgene and transgenic constructs

The {beta}:1-8 transgene has been previously described (13). Briefly, a 4.9-kb BglII genomic fragment of the human {beta}-globin gene (including the {beta}-globin promoter, enhancer, exons, and introns) was linked 5' of a fragment containing HS1–8 of the TCR{alpha} LCR. The hCD2{Delta}T reporter gene has been previously described (19). A stop codon was introduced into exon V of the gene, resulting in a molecule that lacks 116 aa of the cytoplasmic tail normally required for signaling. The hCD2:1-8 transgene was constructed in pBluescript (Stratagene) with a 10.5-kb SalI-BamHI fragment of the hCD2 gene that includes the hCD2 promoter and five exons (second and third introns have been removed) linked 5' of a 10.2-kb SalI-SacI fragment containing TCR{alpha} LCR HS1–8 (13).

RNA analysis

RNA was purified, according to the single-step RNA isolation protocol (23), from mouse organs that were rinsed in PBS (to remove excess blood) and homogenized. RNA was also purified from T and B cells isolated from spleen through magnetic bead separation (described below). Five to 10 µg of each RNA sample was run on a 0.8% agarose gel and transferred onto a nylon membrane for hybridization. A 500-bp EcoRV-PstI fragment from exon II of the hCD2 gene was used as a probe for the transgene mRNA. To detect the human {beta}-globin transgene RNA, a 428-bp BamHI-NcoI genomic fragment of the human {beta}-globin gene coding region was used as a probe. A 0.5-kb Sau3A I fragment from the TCR{alpha} constant region cDNA was used as a probe for endogenous TCR{alpha}. To control for differences in loading and transfer efficiency, a probe to 18S rRNA (Ambion) was used. All probes were labeled with [{alpha}-32P]dATP and/or [{alpha}-32P]dCTP using random primers (Invitrogen Life Technologies). Transgene and endogenous TCR{alpha} mRNA were quantified and normalized to 18S rRNA by PhosphorImager (Molecular Dynamics) analysis.

Flow cytometric analysis

Single-cell suspensions of thymocytes and spleen cells were stained in FACS staining medium (RPMI 1640 medium supplemented with 3% FBS and 10 mM HEPES buffer solution). For each sample, 106 cells/100 µl volume were stained with 0.2–1.0 µg of the respective Abs for 20 min at 4°C. For staining with Abs derived from mouse hybridomas, samples were first blocked for 20 min at 4°C with a 10-fold excess of normal mouse IgG and/or rat anti-mouse CD16/32 (Caltag Laboratories) per 106 cells to reduce background staining. To detect surface expression of hCD2, the following Abs were used: mouse anti-hCD2 from clone S5.2 conjugated to FITC or PE-Cy7 (BD Pharmingen) and mouse anti-hCD2 from clone S5.5 conjugated to PE-Cy5 or PE-Cy5.5 (Caltag Laboratories). For analysis of thymic subsets, rat anti-mouse CD4 (GK1.5) conjugated to FITC and rat anti-mouse CD8{alpha} (53-6.7) conjugated to PE (both from BD Pharmingen) were used. Rat anti-mouse CD44 conjugated to PE and rat anti-mouse CD25 conjugated to allophycocyanin (both from Caltag Laboratories) were used to analyze thymic DN subsets. To analyze spleen subsets, the following PE-conjugated Abs were used: rat anti-mouse CD19 (BD Pharmingen), mouse anti-mouse CD90.2 (Caltag Laboratories), rat anti-mouse F4/80 (Caltag Laboratories), and rat anti-mouse Gr-1 (Caltag Laboratories). PE-conjugated hamster anti-mouse {gamma}{delta} TCR (GL3; BD Pharmingen) was used for analysis of {gamma}{delta} T cells. Flow cytometric acquisition was performed on FACSCalibur (BD Biosciences) for four-color analyses and on FACScan (BD Biosciences) for two- and three-color analyses. Flow cytometry data were analyzed using CellQuest Pro.

Cell purification

The MACS and all kits used for the purification of cell subsets from thymus and spleen were obtained from Miltenyi Biotec. Enriched cells of at least 95% purity were used in experiments. The pan T cell isolation and B cell isolation kits were used to isolate mouse T and B cells, respectively. Rat anti-mouse CD19 and mouse anti-mouse CD90.2 were used to confirm the purity of spleen B and T cells, respectively. To purify DN thymocytes for DN subset analysis, a single-cell suspension of thymocytes in FACS staining medium was first stained with the FITC-conjugated versions of rat anti-mouse CD8{alpha} (53-6.7), rat anti-mouse CD4 (GK1.5), and hamster anti-mouse {gamma}{delta} TCR (GL3; all obtained from BD Pharmingen), followed by removal of labeled cells with an anti-FITC MicroBeads kit (Miltenyi Biotec). For analysis of thymic {gamma}{delta} T cells, DN T cells were isolated as described, except that FITC-conjugated anti-mouse {gamma}{delta} TCR (GL3) was not used in the initial step. Flow cytometry was used to confirm the purity of DN thymocytes by using FITC-conjugated versions of rat anti-mouse CD8{beta} (53-5.8) and rat anti-mouse CD4 (RM4-4), both of which are derived from clones producing Abs recognizing different epitopes than those used for purification. FITC-positive cells were gated out for flow cytometric analysis of purified DN thymocytes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Novel transgenic model for single-cell analyses of TCR{alpha} LCR activity

We have developed a transgenic model, using the hCD2 gene as a reporter, for investigation of LCR activity at the single-cell level using flow cytometry. The hCD2 gene is regulated by an LCR in its natural locus (6). Transgenic mouse studies have demonstrated that hCD2 transgene expression is dependent on the activity of its LCR. Without the hCD2 LCR, the hCD2 transcription unit used in this study is very poorly expressed and is subject to severe site of integration position effects (24, 25). Its LCR dependence makes this hCD2 transgene ideal for use as a reporter of TCR{alpha} LCR function in mice. The hCD2:1-8 transgene construct used in this study consists of the five exons of the hCD2 gene flanked 5' by the hCD2 promoter sequence and 3' by the wild-type TCR{alpha} LCR (including HS1–8 and HS1'; Fig. 1B). In this study a gene encoding a tailless version of hCD2 is used that lacks the sequences required for hCD2 signaling events (19). High level expression of this tailless hCD2 has no effect on thymocyte maturation in transgenic mice. Three independent transgenic mouse lines bearing the hCD2:1-8 transgene construct were analyzed in this study.

Human CD2:1-8 transgene expression is lymphoid organ specific

High level expression of the previously studied {beta}:1-8 transgene (consisting of the human {beta}-globin reporter linked 5' of the wild-type TCR{alpha} LCR; Fig. 1C) was shown to be restricted to the lymphoid compartments of thymus and spleen (13). To examine the tissue distribution of hCD2:1-8 transgene expression, we performed Northern blot analysis of the three hCD2:1-8 lines (4, 29, and 44). Fig. 2A shows the typical expression profile of the hCD2 transgene. Human CD2 mRNA levels were normalized to 18S ribosomal RNA and quantified by PhosphorImager. In all three lines the highest level of expression was observed in the thymus, whereas spleen expression ranged from 13 to 41% of that in thymus (Fig. 2B). In all other organs examined, hCD2 mRNA was either very low (<3% that in thymus) or not detected. These results are in agreement with the mRNA expression patterns observed for the {beta}:1-8 transgene.



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  		FIGURE 2. Tissue distribution of hCD21:8 transgene expression. A, Northern blot analysis of total mRNA from indicated tissues of hCD2:1-8 lines 4 and 44. The hCD2 transgene signal is normalized to 18S rRNA. B, Phosphorimager analysis of Northern blot experiments of a representative individual from each hCD2:1-8 transgenic line (lines 4, 29, and 44). Transgene expression is represented within each line as a percentage of the organ tissue expressing the highest levels. The absence of a bar indicates undetectable expression levels.

 
The relative numbers of intact transgene copies (determined by Southern blot; data not shown) of hCD2:1-8 lines 4, 29, and 44 were determined to be 22, 19, and 21, respectively. Northern blot analysis was used to measure the relative amounts of hCD2 mRNA per transgene copy in each line (data not shown). Among the three lines, the range of hCD2 expression levels per copy in thymus and spleen was within 1.4- and 1.3-fold, respectively, which is well within the range expected for copy number-dependent transgenes driven by this LCR (13) and others (26).

These data indicate that this new hCD2 transgene system faithfully reproduces the TCR{alpha} LCR characteristics that were observed in the context of the {beta}-globin reporter transgene ({beta}:1-8) system previously studied.

TCR{alpha} LCR activity does not restrict transgene expression to T cells

Flow cytometry was used to further investigate the fine cell-type distribution of LCR activity in the hCD2:1-8 lines. We first examined the relative amounts of transgene expression in thymus and spleen. Whole populations of thymocytes and spleen cells were isolated from adult mice and stained with anti-hCD2. Fig. 3 compares hCD2 surface expression in the thymus and spleen of a representative hCD2:1-8 line. Using the mean fluorescence as a measure of surface expression, it was revealed that in all three lines analyzed, hCD2 levels in the spleen were 20–36% of thymic expression. This result is consistent with the relative amounts of hCD2 mRNA in these organs.



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  		FIGURE 3. Flow cytometric analysis of hCD2 transgene expression in the thymus and spleen of an individual of a representative transgenic line (hCD2:1-8 line 44). Cells from whole thymus and whole spleen were stained with anti-hCD2 Ab. Human CD2 expression is represented as a solid line in the transgenic individual and as a dashed line in the nontransgenic littermate control.  

 
The observed lower levels of spleen transgene expression (relative to thymic expression) in previous LCR studies using the {beta}:1-8 model (13), suggested that the transgene was being expressed T cell specifically, because T cells account for approximately one-fourth of all spleen cells. Flow cytometric analysis of the hCD2:1-8 lines revealed that this was probably not the case, because there is no distinct hCD2-nonexpressing population in the spleen (Fig. 3). To more closely investigate hCD2 expression in the spleen, we used cell type-specific markers to examine hCD2 expression in spleen subpopulations. Anti-CD90 and anti-CD19 were used to distinguish peripheral T cells (Fig. 4A) and B cells (Fig. 4B), respectively. We found that the entire spleen T cell population expresses hCD2. Human CD2 was also detected on the surface of essentially all spleen B cells. Furthermore, variegated expression of hCD2 was detected in subsets of spleen granulocytes and monocytes (distinguished with anti-Gr1 and anti-F/480, respectively; data not shown).



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  		FIGURE 4. Flow cytometric analysis of hCD2 expression in indicated cell types from a representative transgenic individual (hCD2:1-8 line 4). A, Human CD2 expression in CD90+ spleen T cells. B, Human CD2 expression in CD19+ spleen B cells. C, Purified spleen T cells stained with anti-hCD2 and anti-{gamma}{delta} TCR (to identify {gamma}{delta} T cells). D, Purified thymic DN cells stained with anti-hCD2 and anti-{gamma}{delta} TCR. Note that cells in C and D are derived from different individual mice analyzed in separate experiments. Human CD2 expression is represented as a solid line in the transgenic individual and as a dashed line in the nontransgenic littermate control.

 
T cells of the {gamma}{delta} lineage make up only a small percentage of the thymic DN compartment. To examine hCD2 expression in these {gamma}{delta} T cells, we first purified DN cells from the thymus by MACS. Using anti-{gamma}{delta} TCR to distinguish the {gamma}{delta} T cells, hCD2 expression was detected on the surface of the majority of these cells (Fig. 4C). Moreover, hCD2 was also observed in peripheral, spleen {gamma}{delta} T cells (Fig. 4D). These observations are in contrast to an earlier transgenic study of the LCR that demonstrated, by RNase protection assay, the lack of expression of a prerearranged TCR{alpha} reporter in {gamma}{delta} T cells (4) (see Discussion).

The expression pattern of hCD2 transgenes in mice includes B cells (27),. Therefore, we wanted to assess whether the B cell activity we observed was a property of the LCR itself or merely the result of elements on the hCD2 reporter. We used Northern blots to examine transgene mRNA in the B cells of hCD2:1-8 lines and compared this to B cell {beta}-globin expression in a previously studied {beta}:1-8 transgenic line (13). Unlike hCD2, {beta}-globin is not normally expressed in B cells. Transgene mRNA levels within each respective line were measured relative to thymic expression. These experiments revealed transgene expression in the B cells bearing either hCD2 or {beta}-globin reporter gene (Fig. 5). These data indicate that the LCR activity seen in B cells is not solely dependent on hCD2 transgene-specific elements, although hCD2-specific elements probably cause the notably higher (2- to 3-fold) expression relative to that in thymus in the hCD2:1-8 lines than is seen in the {beta}:1-8 line. In any case, it is apparent that the TCR{alpha} LCR, unexpectedly, cannot restrict transgene expression to T-lineage cells.



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  		FIGURE 5. Transgene expression in spleen B cells. A, Northern blot analysis comparing transgene expression in thymocytes and purified spleen B cells of a representative hCD2:1-8 line and a previously described {beta}:1-8 transgenic line. The hCD2 transgene signal is normalized to 18S rRNA. B, Phosphorimager analysis of Northern blot experiments in A. Transgene expression is represented within each line as a percentage of thymic expression.

 
The hCD2:1-8 transgene displays proper TCR{alpha}-like timing of developmental expression during thymocyte maturation

Thymocytes were stained with anti-CD4 and anti-CD8 to distinguish between the thymic developmental stages. In all three lines, a significant increase in hCD2 surface expression was detected at the CD4+CD8+ (double-positive (DP)) stage compared with the more immature CD4–CD8– (DN) thymocytes (Fig. 6). Moreover, this increased expression was sustained throughout thymocyte development. The up-regulation of transgene expression in DP thymocytes is consistent with the extensive rearrangement and expression of the TCR{alpha} gene that occur at the endogenous locus at this stage (28, 29). However, there have been reports of the initiation of TCR{alpha} rearrangement and expression occurring even earlier, during the latter part of the DN stage, DN4 (CD25–CD44–) (20, 22). Human CD2 transgene expression was therefore examined throughout the DN stage by the use of Abs to surface markers CD25 and CD44. DN thymocytes were first isolated from adult mouse thymi by MACS. In the two transgenic lines (lines 4 and 44) examined, we observed that hCD2 expression was activated at DN4, precisely the stage when TCR{alpha} gene rearrangement and expression begin (Fig. 7). This onset of transgene expression under the control of the TCR{alpha} LCR is distinct from the initiation of gene expression under the control of the hCD2 gene regulatory elements, which occurs at an earlier DN stage (DN1) (27). This early expression trait intrinsic to hCD2 may explain the low level/variegating, background transgene expression seen at earlier stages. Nevertheless, these data demonstrate that replacing the hCD2 LCR with the TCR{alpha} LCR changes the developmental timing of linked transgene activation to an endogenous TCR{alpha}-like pattern.



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  		FIGURE 6. Flow cytometric analysis of hCD2 expression in a representative hCD2:1-8 transgenic line (line 44) during thymocyte development. Thymocytes were stained with anti-CD4 and anti-CD8 to differentiate between the thymic developmental stages: DN (CD4–CD8–), DP (CD4+CD8+), and SP (CD4+CD8–). HumanCD2 expression is represented as a solid line in the transgenic individual and as a dashed line in the nontransgenic littermate control.

 


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  		FIGURE 7. Flow cytometric analysis of hCD2 expression during early thymocyte (DN) development. A, Purified DN thymocytes were stained with anti-CD25 and anti-CD44 to differentiate between the DN stages. B, Histograms showing hCD2 expression of a representative hCD2:1-8 transgenic line (line 44) throughout DN development. Human CD2 expression is represented as a solid line in the transgenic individual and as a dashed line in the nontransgenic littermate control. C, Relative mean fluorescence of hCD2 transgene expression during thymocyte DN development in two transgenic individuals (hCD2:1-8 lines 4 and 44), as a percentage of maximum expression. The small percentage of CD4- and/or CD8-positive cells remaining in the purified DN sample was gated out of these analyses by staining with FITC-conjugated anti-CD4 and anti-CD8 Abs recognizing different epitopes than those used in MACS purification.

 
The TCR{alpha} LCR does not support high level peripheral T cell expression

Because, the LCR faithfully reproduces the normal thymic TCR{alpha} expression pattern, we next wanted to examine its activity in mature peripheral T cells. Using anti-CD90 to distinguish T cells from other spleen subtypes, the mean fluorescence of hCD2 staining in these cells was compared with that of thymic T cells. Fig. 8 shows relative hCD2 expression between thymic and spleen T cells of a representative line. Surprisingly, in all lines, hCD2 surface expression was lower in spleen T cells, ranging from 22 to 60% of thymic levels. We wanted to determine whether this down-regulation of hCD2 surface expression is a consequence of decreased transcriptional activity in the periphery. In two of the hCD2:1-8 lines (4 and 44), MACS was used to purify T cells from the spleen, and the levels of hCD2 mRNA in these cells were compared with those in thymocytes from the same animals. A corresponding reduction in hCD2 mRNA levels was seen in the peripheral T cells of both lines (Fig. 9). In line 44, hCD2 transcription was reduced to as little as 46% of thymic levels. A similar result was observed in the {beta}:1-8 line, where {beta}-globin transcription was reduced in spleen T cells to 15% of thymic expression levels. To compare these data to the behavior of the endogenous TCR{alpha} gene in these same samples, the mRNA levels of TCR{alpha} were analyzed on the same blots using a probe to the TCR{alpha} constant region (Fig. 9). In contrast to the down-regulation of hCD2 transcription in the periphery, the endogenous TCR{alpha} gene transcription was increased by as much as 56% in line 4 and by >100% in the {beta}-globin line.



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  		FIGURE 8. Flow cytometric analysis comparing hCD2 transgene expression in thymocytes and spleen T cells of a representative transgenic line (hCD2:1-8 line 44). A, Thymocytes and purified spleen T cells were stained with anti-hCD2 Ab. Human CD2 expression is represented as a solid line in the transgenic individual and as a dashed line in the nontransgenic littermate control. B, Relative mean fluorescence of hCD2 transgene expression in two representative transgenic individuals from each line, as a percentage of thymic expression within the same individual.

 


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  		FIGURE 9. Northern blot analysis comparing thymocyte and spleen T cell expressions of transgene and endogenous TCR{alpha} mRNA. A, Transgene mRNA was analyzed in the thymus and purified spleen T cells of two hCD2:1-8 lines (lines 4 and 44) and of a previously described {beta}:1-8 transgenic line. Blots were stripped and analyzed for endogenous TCR{alpha}. Transgene and TCR{alpha} signals were normalized to 18S rRNA. B, Phosphorimager analysis of Northern blot experiments in A. Expression is represented within each line as a percentage of thymic expression.

 
The reduction of peripheral transgene expression observed in both the hCD2:1-8 and the previously studied {beta}:1-8 transgenes renders unlikely the possibility that a reporter gene-specific effect is causing the unexpected down-regulation. These results suggest the requirement for additional, as yet unidentified, elements at the endogenous TCR{alpha} locus for high level peripheral TCR{alpha} expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Immunologists increasingly rely on transgenic animal models of immune development and function. In addition, gene therapy is emerging as a method for the potential treatment of many diseases, including immunological disorders. Progress in gene therapy will depend in part on the development of vectors that can function predictably in an in vivo chromatin environment. Therefore, it is ever more important to identify and understand the full in vivo requirements for ensuring the proper developmental timing and cell type specificity of the gene expression at the heart of hemopoietic differentiation. The study of LCR activity has significantly contributed to our knowledge of these processes. Well-characterized LCRs have also been very useful in the development of physiological transgenic mouse models. LCRs have been implicated in the regulation of several immunologically important genes. Of these, the most extensively studied is the hCD2 gene (6). Others include the human class I MHC molecule, HLA-B7 (7), the mouse pre-BCR subunits {lambda}5 and VpreB1 (8), the macrophage lysozyme (9), the mouse TCR{gamma} (10), the human T cell-specific adenosine deaminase (11), and the mouse Th2 cytokine cluster (12). Partial LCR activity has been discovered in the mouse IL-2 (30) and mouse IgH (31, 32) gene loci.

We have shown in this study that the TCR{alpha} LCR provides integration site-independent transgene expression that is activated at thymocyte stage DN4. This is the stage when endogenous TCR{alpha} gene rearrangement and expression begin (20, 22). The combination of DN4 stage activation and integration site independence of transgene expression has not been previously reported. Yet, these are precisely the properties required for physiological expression of TCR{alpha} transgenes in mice. The procedures used to date to make prerearranged {alpha}{beta} TCR transgenic mice suffer from the problem of premature TCR{alpha} gene transcription that adversely affects thymocyte development (33, 34). Even V{alpha}J{alpha} knockin mice display premature TCR{alpha} expression (35), probably due to other early-activated transcriptional control elements that exist in the wider TCR{alpha}{delta} locus (2). A recent advance in TCR{alpha} transgene expression was reported, in which a relatively cumbersome cre-loxP transgene strategy is used (36). This system yielded the onset of TCR{alpha} transgene expression in DP thymocytes (somewhat later than endogenous TCR{alpha} activation). Significantly, this report demonstrated that the timing of TCR{alpha} expression in mice has profound effects on T cell developmental processes. Our data indicate that the important goal of straightforward, reliable, and developmentally correct activation of prerearranged TCR{alpha} transgene expression in the thymus may be achieved by simply linking such a transgene to the complete TCR{alpha} LCR. Although reporter transgenes linked to the E{alpha}/HS1 element alone are generally very poorly expressed (4, 37, 38), one previous study did show some transcriptional up-regulation of an E{alpha}-linked TCR{beta} rearrangement substrate in CD4–CD8–CD25– triple-negative (rough DN4 equivalent) stage thymocytes (39). This would suggest that the E{alpha}/HS1 element is a major determinant of the specificity of the LCR’s developmental timing.

Our work now allows the TCR{alpha} LCR to be included among a group of gene regulatory elements that activate expression at defined stages of T cell development. The hCD2 LCR has been shown to activate transcription as early as DN1 (27). The proximal promoter of the lck gene activates transcription in CD44+ T cells before the DN3 stage (40). Under control of the CD4 promoter, proximal enhancer (E4Pro), and silencer elements, gene expression is activated in DP thymocytes, whereas the CD8 enhancer (E8SP) is active in T cells committed to the CD4–CD8+ lineage (41). These elements have all been linked to Cre recombinase to selectively manipulate genes (flanked by loxP sequences) in a stage-specific manner during T cell development. The TCR{alpha} LCR may similarly prove useful for DN4 stage-activated expression of Cre recombinase or other desired proteins to study processes occurring in post-TCR{beta} rearrangement thymocytes.

In the present study we report TCR{alpha} LCR activity in non-{alpha}{beta} T cell lineages, including {gamma}{delta} T cells. An earlier study of the TCR{alpha} LCR reported {alpha}{beta} T cell-specific expression of an LCR-linked, prerearranged TCR{alpha} reporter transgene, driven by a V{alpha}-region promoter (4). In those studies, TCR{alpha} reporter gene transcription was not detected in adult thymic {gamma}{delta} T cells using RNase protection assays. We attribute the difference in our findings to the greater degree of sensitivity afforded by the flow cytometry technique used in this study. Although we do not use a V{alpha} promoter in this study, V{alpha} promoters have been shown to be functionally rearranged into TCR{alpha} and TCR{delta} genes (in {alpha}{beta} and {gamma}{delta} T cells, respectively) (42). Therefore, they would not be likely to confer {alpha}{beta} T cell specificity themselves. With our study, the data from experiments using TCR{alpha} LCR-driven transgenes are now in concordance with data from the endogenous TCR{alpha}/{delta} LCR that indicate a function for TCR{alpha} LCR elements in the {gamma}{delta} T cell lineage. A targeted deletion of the sequences known to contain both E{alpha} (HS1) (43) and HS1' (14) resulted in decreased transcription of the TCR{delta} gene in {gamma}{delta} T cells (44). Also, DNase I hypersensitivity assays performed in primary {gamma}{delta} and {alpha}{beta} T cells revealed the formation of endogenous HS1 and HS1' in both cell types (14).

Past whole organ analyses have also suggested that TCR{alpha} LCR activity was restricted to T-lineage cells. Subsequent transgenic studies have detected B cell expression in transgene configurations involving TCR{alpha} LCR elements. However, these studies used either indirectly linked, cointegrated TCR{alpha} LCR (45) or incomplete TCR{alpha} LCR cointegrated with a TCR{beta} transgene (46). We directly addressed this question at single-cell resolution in a directly linked, complete LCR-driven reporter transgene system free of integration site-dependent position effects. We definitively show the expression of TCR{alpha} LCR-linked transgenes in cells of the B lineage. Because studies of hCD2 transgenes have shown its expression in the mouse B cell compartment (27, 47), we were concerned that the ectopic B cell expression seen in our study may be an artifact of the hCD2 reporter. To address this concern, we examined the pattern of a TCR{alpha} LCR-linked human {beta}-globin reporter fragment and still observed expression in B cells. It is worth noting that the purified {beta}-globin transgenic B cells used in Fig. 5 were 99% CD19 positive (data not shown). Furthermore, spleen T cell {beta}-globin expression in this line is also fairly low relative to thymic levels (Fig. 9). This renders unlikely the possibility that the {beta}-globin mRNA expression seen can be explained by T cell contamination. Because {beta}-globin is not normally expressed in B cells, we conclude that the LCR must have some activity in B cells. A higher level of B cell expression for the hCD2 reporter is probably observed because its elements are better adapted to generating expression in B cells under the influence of the LCR than is the {beta}-globin reporter. Nevertheless, it is clear that the TCR{alpha} LCR elements, although sufficient to direct expression to lymphoid organs, are unexpectedly insufficient in restricting linked transgene activity to T-lineage cells.

The above data concerning lineage specificity raises the essential question of how the cell type restriction of the ultimate gene products of the endogenous TCR{alpha}{delta} locus (TCR {alpha}- and {delta}-chains) is achieved. Given that transcripts from the endogenous TCR{alpha}{delta} locus are not normally observed in B cells, it is highly likely that other gene regulatory elements in the wider TCR{alpha}{delta} locus outside the LCR are required to restrict transcription from this locus to T-lineage cells. Our data provide a rationale for further investigation of this possibility. The presence of other gene regulatory elements (apart from the LCR) in the TCR{alpha}{delta} locus also lends support to this idea (48). The case of restriction to {alpha}{beta} vs {gamma}{delta} lineage T cells is less clear. Both selective transcriptional (4, 49) and selective rearrangement (39, 50) mechanisms have been invoked to explain the ultimate cell type restriction of TCR{alpha} and TCR{delta} protein production. Yet, rearrangement of TCR{delta} loci are seen in T cells destined to become {alpha}{beta} T cells (51) and TCR{alpha} locus-derived transcripts are observed in {gamma}{delta} T cells (44). Although our experiments do not address rearrangement, our studies now bring the data on TCR{alpha} LCR-driven transgenes into concordance with the idea that lineage-selective gene rearrangement mechanisms may be of greater importance in {alpha}{beta} vs {gamma}{delta} T cell subset restriction than transcriptional mechanisms per se.

Finally, we report that the TCR{alpha} LCR loses potency (in sharp contrast to the up-regulation of endogenous TCR{alpha} transcription) as T cells transit from the thymus to peripheral lymphoid organs. These observations suggest the unexpected existence of differential mechanisms of TCR{alpha} gene regulation in the thymus and peripheral T cells. Similar observations have been described in other developmentally regulated lymphocyte gene loci, such as mouse CD8 (52, 53), mouse CD4 (54), mouse lck (55), and, in B cells, the mouse IgH locus (56). This recurring finding of distinct thymic and peripheral gene regulatory mechanisms in T cells may relate to a need for thymic transcriptional mechanisms to be sensitive to signaling input occurring during thymic selection and/or V-D-J recombination processes.

Our data indicate that all the elements required to direct the correct developmental timing of TCR{alpha} expression in the thymus are contained within the LCR. However, it is apparent that additional elements from the wider TCR{alpha}/TCR{delta}/Dad1 locus must participate in conferring fine cell type specificity. It is also evident that other gene regulatory mechanisms not supported by the LCR sequences are necessary to up-regulate TCR{alpha} gene expression in peripheral T cells. It will be important to identify these mechanisms and the transcriptional, or perhaps post-transcriptional, control elements that govern them. Reporter transgenes that incorporate wider regions of the natural TCR{alpha} gene locus, such as is possible with bacterial artificial chromosomes, will be required to pursue these interesting lines of investigation.


   Acknowledgments
 
We thank Joon Kim for expert flow cytometry assistance, the Rockefeller University Transgenic Service Laboratory for the generation of transgenic mice, and Anna Kloc, Martina Levisohn, and Karl Erhard for technical assistance. We also thank Rose Zamoyska and Dimitris Kioussis for generously providing the hCD2 reporter gene, and Derek Sant’Angelo for helpful comments and critical reading of this manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health (NIH) Grant AI053050 (to B.D.O.) and National Science Foundation Career Award MCB0236964 (to B.D.O.). This work was begun with startup funding from the Support for Continued Research Excellence program funded by the NIH Grant GM60654 (to Hunter College). Support of the infrastructure and instrumentation in the Department of Biological Sciences at Hunter College was provided by Research Centers in Minority Institutions Award RR03037 from the National Center for Research Resources, NIH. F.H. was supported by NIH Grant GM060665 for the NIH Research Incentive for Scientific Enhancement program at Hunter College and is the recipient of a Ford Foundation Dissertation Fellowship from the National Research Council. Back

2 Address correspondence and reprint requests to Dr. Benjamin D. Ortiz, Department of Biological Sciences, City University of New York, Hunter College, 695 Park Avenue, Room 927-N, New York, NY 10021. E-mail address: ortiz{at}genectr.hunter.cuny.edu Back

3 Abbreviations used in this paper: Dad1, defender against death-1; DN, double negative; DP, double positive; HS, DNase I-hypersensitive site; h, human; LCR, locus control region. Back

Received for publication May 20, 2005. Accepted for publication September 6, 2005.


   References
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
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