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The Generation of Human T Cell Repertoires During Fetal Development¹

Laila D. McVay^*, Sheila S. Jaswal^2,*, Christine Kennedy^3,*, Adrian Hayday and Simon R. Carding^4,*

^* Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and Department of Biology, Yale University, New Haven, CT 06520

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The nature of how human T cells are normally generated is not clear. We have used an RT-PCR assay and DNA sequencing to identify and compare -encoded TCRs (TCRDs) that are generated de novo in the fetal gut, liver, and thymus and to determine when, where, and how the TCRD repertoire is established during normal embryonic development. Rearranged TCRDV genes are first expressed outside of the thymus in the liver and primitive gut between 6 and 9 wk gestation. Although DV1Rs and/or DV2Rs predominated, differences in the pattern of TCRDV gene rearrangement and transcription in each tissue during ontogeny were identified. Specific, DV2-encoded TCRs are highly conserved throughout ontogeny in the tissues from the same and between genetically distinct donors. Although the thymic and intestinal T cell repertoires partially overlap early in development, they diverge and become nonoverlapping during the second trimester, and the generation of the intestinal T cell repertoire is characterized by differences in the processing of DV1Rs and DV2Rs. Whereas the structural diversity of DV1Rs progressively increases during gut development up to birth, DV2Rs have limited structural diversity throughout ontogeny. Together, our findings provide evidence for the ability of different fetal tissues to support the development of T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The nature of how the human T cell repertoire is generated is poorly understood. Unlike ß T cells, which are almost exclusively dependent upon the thymus for their development, T cells can be generated in extrathymic sites. Recently, we have shown that the fetal liver is an extrathymic site for T cell development during normal human development (1). CD3⁺ lymphocytes that express TCRs⁵ can be identified before thymic development at 6 to 8 wk gestation. These cells express TCRs that are essentially monomorphic and capable of responding to microbial antigenic stimulation. Other studies that have identified populations of T cells unique to the liver are consistent with our finding that the liver is a site of T cell development (2, 3). One question raised by these findings is whether other fetal hematopoietic tissues, such as the fetal intestine, are also sites of human T cell development; if so, are the populations generated distinct from those produced in the fetal liver and later in the thymus?

A comparative analysis of TCRDV gene usage by T cells (4) in the fetal and adult gut has shown they are distinct. TCRDV2⁺ T cells have been shown to predominate in the fetal gut (5), whereas TCRDV1⁺ T cells have been shown to be the major T cell population in the adult human intestine, comprising 30% of intestinal intraepithelial lymphocytes (IELs)⁶ (6, 7, 8). Interestingly, unlike the adult human thymus, the adult intestinal TCR- and TCR-ß repertoires are oligoclonal (9, 10, 11, 12, 13, 14). Recent studies suggest that this oligoclonality is established between neonatal and adult life (15). However, how this oligoclonality occurs is currently not known.

The intestinal epithelia, similar to the thymic epithelium, may be able to support T cell development, and several lines of evidence suggest a lymphopoietic capacity of the murine small intestine. Within the epithelial spaces, cells can be identified that express markers that are characteristic of immature T cells, such as the CD3^-CD4^-CD8⁺ cells which are TCR^- but contain abundant recombination activating gene (RAG) transcripts (16). Unique structures in the crypts known as cryptopatches contain cells that are phenotypically immature (17). Ectopically transplanted fetal intestinal grafts in athymic mice have been shown in some studies (18) to support T cells within the grafts and the generation of T cells in the peripheral compartment. Positive and negative selection of murine intestinal IELs (19, 20, 21) has been shown, demonstrating a capacity of the intestinal epithelium to provide a suitable milieu for selection. Finally, the ability of intestinal epithelial cell lines to promote the phenotypic maturation of T cells during in vitro culture suggests that the intestinal epithelia may be able to induce or support the differentiation of T cell progenitors (22, 23, 24).

Despite these studies, the possibility that the human fetal intestine can support T cell development has not been addressed. To understand more clearly how the T cell repertoire is generated at the molecular level and the nature of the mechanisms that might contribute to their selection and survival, we have conducted a detailed ontogenetic analysis of TCR gene expression in the intestine, comparing it with that seen in the liver and thymus during human fetal development. We have used a large number of samples from various ages during fetal development to analyze the expression of TCRDV genes in the fetal liver and gut before thymic colonization; this expression was also analyzed in the thymus, liver, and gut after thymic colonization from the same donors when possible. We provide evidence for the ability of different fetal tissues to influence the development of human T cells and identify features of the TCRDV intestinal repertoire that distinguish it from those expressed in other fetal tissues.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue samples and cell isolation

Fetal tissue samples were procured by the Anatomic Gift Foundation (Laurel, MD) with the approval of the University of Pennsylvania Committee for Studies Involving Human Subjects (Philadelphia, PA). The gestational age of individual samples was determined according to the Carnegie embryonic staging system (25, 26). Where possible, microscopic examination was also used to establish the absence or presence of a thymic rudiment. The liver, thymi, and intestine samples obtained from same donors and used for this study were: 6 wk: No. 6-01 (liver, gut); 6.5 wk: No. 6.5-48 (thorax, liver, gut); 8.7 wk: No. 8.7-691 (thymus, liver, gut); 9 wk: No. 9-14330 (thymus, liver, gut), No. 9-14449 (thymus, liver, gut), and No. 9-32043 (thymus, liver, gut). The origin and age of other samples used in this study is shown in Table I.

RT-PCR analysis of the TCRV repertoire

Total cellular RNA was prepared from fetal tissue as previously described (27). RNA pellets were extracted sequentially with acidic-phenol (once), phenol-chloroform (1:1, once), and chloroform (twice) before ethanol precipitation. We used 10 µg of total cellular RNA to synthesize cDNA with random hexanucleotides. The amount of cDNA synthesized from each RNA sample was normalized according to the amount of human ß-tubulin PCR product generated from 1 µl of cDNA that had been amplified using ß-tubulin-specific primers (28). The primers and the RT-PCR assay used for the TCRDV gene segment analysis have been described previously (27). Briefly, oligonucleotide primers specific for DV1, DV2, or DV3 in combination with a DC gene segment primer were used to amplify cDNA in a 10 µl reaction volume using an air-driven thermal cycler (Idaho Technology, Idaho Falls, ID). A nested PCR assay was used to analyze the DV-DJ usage (1). The products of DV-DJ amplification were electrophoresed, and TCRD PCR products were gel purified and quantitated by DNA fluorometry. Equivalent amounts of each cDNA were used as a template for a second amplification using the same forward DV primer and a reverse primer that was specific for each of the four human DJ genes. The reverse primers corresponding to the DJ gene segments used for the analysis of DJ gene usage among amplified DV1-DCRs, DV2-DCRs, and DV3-DCRs have been described previously (1). For DV/DJ gene analysis, DV1, DV2, and DV3 oligos that lie at the 3' end of each DV gene and internal to the priming oligos were used as probes. Individual DV1-DJ1–4, DV2-DJ1–4, and DV3-DJ1–4R cDNA clones were used to determine the optimum conditions for the proportional amplification of DV/DJ genes without cross-priming.

mAbs and flow cytometry

The murine mAbs reactive with human TCR- and CD8 proteins were purchased from T Cell Diagnostics (Boston, MA). The anti-human CD3 Ab was obtained from Becton Dickinson (Sunnyvale, CA). The anti-human ß Ab, BMA031, was obtained from Behringwerke AG (Marburg, Germany). Before staining, fetal liver and gut mononuclear cell (MNC) preparations were depleted of erythroid cells by immunomagnetic depletion (28) using an anti-human glycophorin A-specific Ab (Immunotech, Westbrook, ME); the remaining 10 to 15% of cells were incubated with Abs to human FcR II (clone 4.3, American Type Culture Collection (ATCC), Manassas, VA) and FcR III (clone 3G8, ATCC) to block the nonspecific binding of fluorochrome-conjugated Abs. For the two-color flow cytometric analysis of fetal T cells, between 2 x 10⁵ and 1 x 10⁶ cells were initially stained with biotinylated primary Abs and then stained with phycoerythrin-conjugated Abs and streptavidin-Red613 (Life Technologies, Gaithersburg, MD) as described previously (29). Stained cells were run on a FACScan (Becton Dickinson) and analyzed using Lysis II software.

DNA sequencing

PCR-amplified DV1-DCR and DV2-DCR cDNAs were agarose-gel purified (Glas Pac/GS, National Supply Company, San Rafael, CA) and cloned into the pGEM-T vector (Promega, Madison, WI) or the TA vector (InVitrogen, San Diego, CA). Plasmid minipreps from randomly picked colonies were used to obtain DNA for the sequencing of both strands by the dideoxynucleotide chain termination method using modified T7 polymerase (Sequenase Version 2.0, U.S. Biochemical, Cleveland, OH).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
An overview of human fetal gut development

The primitive gut forms during wk 4 of gestation as the head, tail, and lateral folds incorporate the dorsal part of the yolk sac into the embryo. The primitive gut is divided into the foregut, the midgut, and the hindgut, which can be distinguished on the basis of arterial blood supply; the celiac, superior mesenteric, and inferior mesenteric arteries vascularize the foregut, the midgut, and the hindgut, respectively. Branches of the celiac artery vascularize outgrowths of the foregut, in particular the liver, early during wk 4, establishing a common blood supply and a link between the developing gut and liver (30). This finding has important implications in understanding the origin of the first lymphoid cells. Intestinal IELs have been identified at 11 wk (31), and lymphocytes in the lamina propria have been identified at 14 wk (32). Peyer’s patch aggregates are first evident at 11 wk; these aggregates are diffusely colonized with T and B lymphocytes at 16 wk and with defined zones of the germinal follicle of Peyer’s patches at 19 wk (33), suggesting that the anatomic organization of the fetal intestine is apparent during the first trimester.

Study outline and samples

Our study was designed to address how the human T cell repertoire is normally generated in vivo during fetal development. Specifically, we addressed when and in which tissues TCRDV-DC genes are first activated, whether the fetal gut is a site of T cell development, and, if so, whether the populations generated are distinct from those found in other sites of T cell development, such as the fetal liver and thymus.

We focused on the analysis of T cell development in the fetal intestine, liver, and thymus before and after thymic development and colonization by T cell precursors. TCR gene expression was restricted to the analysis of the TCRDV repertoire since, in contrast to TCRGV mRNA expression (34), TCRDV mRNA expression correlates well with protein expression. Although six DV genes have been described (35), subsequent analyses have shown that TCRDV4, TCRDV5, and TCRDV6 are TCRA genes (TCRAV6.2, TCRAV21, and TCRAV17, respectively) (35, 36, 37, 38). Since rearranged TCRDV1-, TCRDV2-, and TCRDV3-DC genes preferentially rearrange to DJ gene segments to form TCRDRs rather than to AJ gene segments to form TCRARs, our study was restricted to these three predominant TCRDV-DJ-DC genes.

Since the quantities of fetal tissues obtained were small, and cell numbers were low, it was not possible to routinely perform analyses on isolated TCR⁺ cells. Instead, an RT-PCR assay was used to characterize TCRDVR transcription. The specificity of the primers used was verified using individual TCRDV-DC cDNAs cloned from T cell lines or clones. No PCR product was amplified in the absence of cDNA or when an irrelevant cDNA (fetal kidney) was used as a substrate. Over a concentration range of 10⁴-fold, the amount of PCR product obtained was proportional to the amount of substrate cDNA as detected by Southern blot hybridization. This finding was determined by the amplification of a range of concentrations of T cell cDNA diluted into fibroblast cDNA as described previously (28). This assay cannot, however, provide any quantitative data concerning TCRDV gene expression, and the conclusions reached in this study rely on detecting changes or differences in the relative levels of TCRDV gene expression.

To identify and analyze TCRDVR transcription that may occur independently of any thymic influence, liver and gut samples were obtained before thymic colonization at 6 wk gestation. In addition, thorax/thymus, liver, and gut samples were obtained before or coincident with thymic colonization at 8 wk gestation. In some cases, it was possible to obtain multiple tissues from the same donor. To overcome the potential variability of human tissue and to analyze TCRDV gene rearrangement at later times during fetal development, >100 additional tissue samples were used (Table I).

TCRD gene rearrangement and transcription in the fetal liver and gut before thymic colonization

Our first indication that TCRDVR transcripts might be activated in the fetal liver and gut independently of the thymus was detecting expression of RAG-1 and RAG-2, which are normally required for the rearrangement of TCR genes (39, 40), in the 6 wk liver and gut (Ref. 1 and data not shown). Consequently, fetal liver MNCs and gut tissue mRNA samples between 6 and 9 wk gestation were analyzed for evidence of TCRDV-DJ using a sensitive RT-PCR technique that had been previously optimized for the analysis of TCRGV and TCRDV genes in the human fetal thymus (27) and liver (1), as described in Materials and Methods.

Although the profile of TCRDVR transcripts in the liver and gut were very similar at 6 wk, there were differences in the extent of DJ usage in TCRDV1-, TCRDV2-, and TCRDV3-encoded receptors (Fig. 1). Whereas TCRDV1R (Fig. 1A) and TCRDV2R transcripts (Fig. 1B) almost exclusively used DJ1 and/or DJ3 genes, all of the DJ gene segments were used in DV3R transcripts (Fig. 1C). In the 8 wk samples, it was possible to identify profiles of TCRDV-DJR transcripts that were unique to each tissue. In the gut, the restricted use of DJ1and DJ3 in TCRDV1-encoded receptors (Fig. 1A) and the use of all DJ gene segments in TCRDV3R transcripts (Fig. 1C) distinguished it from the profiles seen in the thymus and liver. In addition, the predominance of DV3-DJ3R transcripts in the 8 wk liver distinguished it from the thymus, in which TCRDV3R-transcripts were barely detectable (Fig. 1C).

View larger version (43K): [in this window] [in a new window]   FIGURE 1. TCRDV-DJ gene expression in fetal hematopoietic tissues before (6–8 wk) and immediately after (9 wk) thymic colonization. cDNA samples from fetal liver and thymic MNCs or primitive gut tissue mRNA were amplified using DV1 (A), DV2 (B), or DV3 (C) and DC-specific primers. Aliquots of the DV-DC PCR products were then reamplified by nested PCR using the same DV primer and a reverse primer that was complimentary to each of the known DJ genes. Amplified DV-DJ PCR products were detected by Southern blot analysis using a DV-specific primer that was internal to the DV PCR primer as a probe. Control PCR samples contained either no cDNA (A–C) or fetal kidney cDNA (A). Autoradiography lasted for 1 day. The first digit of the sample code number represents the gestational age in weeks, and the hyphenated number represents the code number used to identify each sample.

To extend this analysis and determine whether and how the TCRDV repertoire changes later in fetal development, an additional 117 fetal tissue mRNA samples obtained from donors between 9 and 33 wk gestation (Table I) were analyzed. The Southern blot analysis of PCR-amplified TCRDV-DC genes from most of these fetal tissues is summarized in Table II.

The pattern of TCRDVR transcripts changes in conjunction with thymic colonization (Table II). TCRDV1 predominated between 15 and 16 wk, while the expression of the other TCRDV genes was only detected after a long (4-day) exposure. TCRDV1 and TCRDV2 were more strongly expressed than TCRDV3 transcripts between 17 and 22 wk and at 33 wk gestation, with TCRDV2 being predominant. Within the postnatal thymus, DV1 expression was predominant, and DV2 expression could not be detected, as we (1) and others (41, 42, 43, 44) have previously observed.

The pattern of TCRDVR transcripts also changed during fetal liver ontogeny and was distinct from that seen in the thymus (Table II). Whereas most DV transcripts were detected between 9 and 14 wk in the thymus, the profile of transcripts in the liver was more restricted, with DV2 predominating in the majority of samples analyzed. Indeed, DV2 was the only transcript detected in five of six 16 wk samples. At 17 to 22 wk, all DV transcripts were detected, with DV2 remaining predominant.

In the fetal intestine, most TCRDVR transcripts were expressed throughout ontogeny. However, in contrast to the liver and thymus, a single receptor, DV2, was predominant throughout ontogeny (Table II). DV1 expression was more variable; this expression was predominantly seen at 9 to 11 wk, 15 to 15.5 wk, and 33 wk. The apparent increase in the amount of DV2 transcripts detected as ontogeny proceeds (Table II and data not shown) may reflect an increase in the number of TCR-⁺ cells or in the number of transcripts per cell. This increase is coincident with the time in gut development during which it is possible to isolate sufficient numbers of T cells for analysis (see below).

Summary of the TCRDVR transcription pattern in the thymus, liver, and gut

While it is not surprising that there is some variability between age-matched samples, consensus patterns of expression were apparent (Table II). For example, one striking feature was the singular predominance of DV1R transcripts in the 15 to 16 wk fetal thymus and DV2R transcripts in the 16 wk fetal liver. Also, the overall pattern of DV3 transcripts was less variable than DV1 or DV2 transcripts in all of the samples examined. DV1 was predominant in both the 3-yr postnatal thymus and the adult intestine, as previously described (9, 10, 27, 41, 42, 43) and is included here to demonstrate the comparison of TCRDVR transcripts in fetal and adult tissues.

Structure of TCRDV1R transcripts expressed before and after thymic colonization

Although the normal adult intestine is characterized by the oligoclonality of the TCR- repertoire (9, 10, 11, 12, 13, 14) that becomes apparent after birth at between 14 and 17 years of age (15), the analysis of oligoclonality in fetal development had only been studied at a single timepoint (20 wk) (15). Thus, we were interested in determining whether any oligoclonality was apparent during early fetal life, mid-gestation, or late gestation. Therefore, the structure of PCR-amplified TCRDVRs expressed in samples of different gestational ages was analyzed. Since DV1 was the predominant DV gene expressed in the fetal thymus after colonization as well as in the postnatal thymus and the adult intestine (9, 10, 27, 41, 42, 43), this receptor was initially chosen for DNA sequence analysis. In addition, since we have shown here that DV2R transcripts can be detected as early as 6 wk in the primitive gut (Fig. 1B), this receptor was also chosen for DNA sequence analysis.

Table III is a compilation of DV1-DJ and DV2-DJ DNA sequences from the paired 6 wk liver/gut (No. 6-01L, No. 6-01G) and 8 wk thymus/liver/gut (No. 8-691T, 8-691L, 8-691G) samples. Since it was difficult to isolate DV1Rs for sequence analysis in these very early samples (Table IIIA) with a single DV-DC PCR amplification, the PCR products were gel-purified, quantitated, and reamplified in a nested PCR using DV and DC oligos that were lying internal to those used for the initial amplification reaction. Despite this additional amplification step, it was only possible to isolate a small number of cDNA clones from the 6 wk samples; this lack of isolation reflects the small size (1% of liver MNC) (1) of TCR- mRNA-expressing cells in these early fetal tissues. In addition, we were unable to obtain any DV cDNA clones from the 6 wk fetal liver sample. In the 6 wk gut, two distinct receptors were identified; they comprised one or more DD gene segments and primarily used a DJ1 gene segment. These features are uncharacteristic of "fetal" receptors that usually have a single TCRDD gene segment and limited junctional diversity. Neither of these receptors were found in the fetal intestine, thymus, or liver at later stages of gestation.

A comparison of the predicted amino acid sequence for TCRDV1- and TCRDV2-encoded receptors clearly shows that the complementarity-determining region (CDR)3 of the DV1Rs is longer than the DV2Rs (Table IV), further illustrating the difference between DV1R and DV2R transcripts in 6 to 9 wk fetal tissues.

We chose to analyze DNA sequences in 15 wk intestinal samples, since our analysis of TCRDV mRNA expression (Table II) identified a striking change in TCRDV gene expression, particularly in the intestine at this time point, and sufficient numbers of cells for phenotypic analysis could be isolated. At this point in development, the structural diversity of DV1Rs expressed in the intestine was comparable with those expressed earlier during ontogeny (Table IVA). Of the 15 DNA sequences obtained, 10 were productive and were represented only once or twice (Table IVA). This diversity in receptor sequences was attributable to N/P nucleotide insertion and exonucleolytic nibbling, particularly at the 5' and 3' ends of the DD3 gene segment (data not shown). The CDR3 lengths of these receptors were comparable with those expressed in the 6 and 8 wk intestine (Table IVA). Thus, there is no evidence of TCRDV1 oligoclonality in the fetal intestine at this time point.

A DNA sequence analysis of 24 DV1Rs from the 33 wk gut sample demonstrated that the diversity of this receptor repertoire was increased further. Of the 24 sequences obtained, none was represented more than once, and they resembled the structure of neonatal/adult receptors, with multiple DD gene segment usage and N/P nucleotide insertions present (data not shown). A total of 13 of 24 DV1Rs were in frame (Table IVA) and further illustrate the increase in the CDR3 length of these receptors at this time.

The structure TCRDV2Rs in the fetal gut between the first and third trimester of gestation

To determine whether the structural complexity of DV2R genes was similar to DV1R genes and whether the oligoclonality of the DV2 repertoire was apparent at all during fetal life, we analyzed the DV2R genes from the 6 (No. 6-01), 8.7 (No. 8.7-691), 15 (No. 15-185G), and 33 wk (No. 33-01G) gut samples that had been used for the analysis of DV1Rs.

Of the DV2 cDNAs sequenced from the 6 and 8.7 wk gut, the same sequence and receptor was predominant in each sample (Table IVB). Of the DV2 cDNAs sequenced from the 15 wk gut, 12 of 16 were productive, of which two represented approximately half (6 of 12) of those sequenced. Compared with DV1Rs, the DV2Rs were structurally simpler, with limited N/P nucleotide insertion and exonucleolytic processing and use of a single DD gene segment. The CDR3 lengths of 33 wk DV2Rs (Table IVB) were strikingly similar to those identified in the 15 wk sample. In addition, while there is a predominance of DV2Rs using the DJ1 gene segment in the first trimester, this changes to a predominance of DJ3 use in the third trimester. Thus, while DV1Rs between 6 and 33 wk were extensively modified and structurally diverse, DV2Rs in the same donors were fetal in structure.

Thymic and intestinal T cells are phenotypically distinct and use structurally distinct, nonoverlapping TCRDV2Rs

Since the expression of TCR and associated differentiation Ags (CD2, CD3, CD4, and CD8) may distinguish between populations of fetal T cells, a phenotypic analysis of intestinal and thymic T cells that had been isolated from a single 15 wk donor was performed. This analysis could not be performed on earlier samples, because it was not possible to isolate sufficient numbers of cells. Thymocytes and gut lymphocytes isolated from the same 15 wk donor were stained using Abs directed against TCR, CD3, and CD8 cell surface glycoproteins (Fig. 2). T cells comprised 2% of CD3⁺ thymocytes and were CD8^- (and CD4^-, data not shown). The fetal gut MNCs were uniformly positive for CD45 (>95%), variably expressed CD2 (40–50% positive) and CD7 (60–75%), and were essentially negative (<5%) for the myelomonocytic and B cell lineage markers CD11b and CD19, respectively (data not shown). Of these cells, 8 to 9% were CD3⁺, of which virtually all cells were ⁺ (Fig. 2). In contrast to TCR-⁺ thymocytes, intestinal T cells were uniformly CD8⁺. Thus, cells comprise the vast majority of those T cells in the 15 wk gut that can be distinguished from the thymic population on the basis of CD8 expression. Our data does not, however, enable us to establish when CD8 expression is acquired by intestinal T cells and whether it is restricted to cells residing in the intestinal epithelium or is acquired by CD8 thymic migrants that populate the intestine.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Human fetal thymic and intestinal T cells are phenotypically distinct. Thymocytes and intestinal MNCs from a single 15-wk donor were stained with anti-CD3/TCR and CD8-specific Abs and analyzed by flow cytometry as described in Materials and Methods. The level of staining with fluorochrome-labeled, isotype-matched Abs of irrelevant specificity was used to determine the frequency of the positive cells shown in each quadrant of the contour plots.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The expression of RAG mRNA and TCRDVR transcripts in the fetal gut before thymic organ formation and colonization and by lymphocyte progenitors is consistent with the observation that the gut, like the fetal liver (1), is an extrathymic site of de novo gene rearrangement. However, the exact sequence of TCRD gene rearrangment and expression, the site(s) in which it is initiated, and the mechanism by which the gut might be seeded with progenitors are not known. Although we can exclude the thymus as the site of origin of cells expressing the TCRDVR transcripts that are present in the 6 wk primitive gut, our studies cannot discriminate between the following possibilities: 1) that these cells originated in the liver and traveled to the gut, 2) that they were generated in the gut and seeded to the liver, or 3) that they were generated independently in each tissue. The identification of and IEL progenitor populations in the human intestine would help resolve this issue. Although putative IEL progenitor cell populations have been identified within distinct clusters of cells or cryptopatches (17) in the murine intestinal mucosa, analogous populations of cells have not been found in the human intestine to date.

The striking diversity of TCRDV1Rs expressed in the 6 wk primitive gut distinguishes the R expression in this site from that seen in either the thymus or liver at any time during fetal development. The structure of these receptors resembles that seen in adults, suggesting that they may be of maternal origin. The inability to detect terminal deoxynucleotidyltransferase activity in fetal tissues until 12 wk gestation (45) and the presence of V1⁺ T cells within the placental decidua at this time during fetal development (46) is consistent with this possibility. However, if DV1-expressing T cells can cross the placental barrier and gain access to the fetal circulation, this action would be a property that is unique to this subset of T cells, since we have been unable to obtain any evidence for the presence of maternally derived DV2-expressing T cells in fetal tissues (1).

Distinctive patterns of TCRDVR transcripts and receptor structure in different tissues of the same donor may reflect the trafficking of different DV⁺ populations to and from distinct anatomical sites at different times during development. The establishment of a common blood supply between the liver and intestine after wk 4 of gestation and the vascularization of the thymus after 8 wk gestation (30) provides a means by which cells generated in the liver or gut can transit between each tissue and to the thymus. The expression of adhesion molecules by populations or their progenitors promoting their adherence to, and retention by, various stromal cell populations provides a possible mechanism for the distribution and retention of specific cells in different tissues. Indeed, the preferential localization of V1⁺ cells to intestinal epithelia is associated with the expression of a particular integrin, E-cadherin, (47). In addition, we and others have also shown that among human T cells, V1⁺ cells can preferentially adhere to fibroblasts normally found in the skin (48) and colon (49). An alternative but not mutually exclusive explanation for these differences in tissue TCRDV profiles is that they may be a consequence of the selective survival, expansion, or death of particular V-expressing cells by various factors (e.g., Ags and cytokines) that are present in the microenvironments of each tissue.

The conservation of identical, particularly DV2, receptors is another striking feature of human T cell development. In addition to the DV2Rs first detected in the 6 wk fetal liver and gut, receptors with the same sequences can be found among a panel of T cell clones derived from the 14 wk fetal liver (1) and among cDNA clones from the thymus and peripheral blood of adults (50). Although the nature of the mechanisms responsible for this bias in receptor usage are not known, it seems likely that both intrinsic (genetic) or extrinsic (environmental) factors are involved. An analysis of rearranged DV/DC genes on both chromosomes of human peripheral blood, thymus, and intestinal T cell clones has suggested that preferential gene rearrangement accounts for the bias in TCRV region usage in human T cells (51). However, we are unable to exclude the possibility that once the cells expressing these receptors are generated, their survival and growth ("selection") may be dependent upon Ag; the identity of these Ags has not been established. Specific fetal receptors could also be maintained throughout life as a consequence of their continuous generation and/or longevity. The continuous de novo generation of fetal receptors seems less likely, since the mechanisms that diversify AgRs also become more active and are extensively used to generate diverse repertoires as ontogeny proceeds. Their longevity may be due to continuous or periodic exposure to Ag. For example, the expansion of DV2R-bearing T cells that is seen in the peripheral blood of neonates (52, 53) suggests that the initial exposure to environmental Ags is one such timepoint during which fetal or neonatally derived T cells can be expanded. The possibility that the retention of T cell subsets in specific tissue microenvironments may also contribute to their longevity is suggested by the finding that the survival of Ag-stimulated T cells is increased as a result of their ability to bind and interact with tissue fibroblasts (54).

Our comparative analyses of TCRDVR expression in fetal hematopoietic tissues clearly distinguish the intestinal repertoire from that of the liver and thymus. In addition, our results also show that the oligoclonality of TCRDVRs, which is a hallmark feature of the intestinal T cell repertoire in humans (9, 10, 11, 12, 13, 14, 15), is not established until very late (after 33 wk) in gestation or, as suggested by others (15), after birth. Although experiments conducted in conventionalized germfree mice suggest that exposing mucosal T cells to commensal gut bacteria can influence the repertoire of Ag and TCR specificities present (55), and that DV1-expressing human cord blood T cells can proliferate in vitro in the presence of certain bacterial Ags (56), there is no compelling evidence that this mechanism "shapes" the intestinal TCRD repertoire in humans.

A comparison of TCRDV1R and TCRDV2R sequences in 15 wk fetal thymus and gut samples from the same donor indicated that there were no overlapping sequences shared between these sites; the phenotype of intestinal and thymic T cells is distinct and is consistent with functionally distinct receptor-bearing cells residing in different compartments. In addition, a comparison of DV1R and DV2R sequences present in the gut of the same donor at mid-gestation (15 wk) and late gestation (33 wk), showed that each receptor was processed differently, resulting in receptors that have extensive (DV1) or minimal (DV2) receptor sequence diversity. These differences in the processing of gut DV1Rs and DV2Rs may be attributable to differences in the availability or accessibility of each TCRDR to the machinery responsible for diversifying AgRs, such as exonucleases and terminal deoxynucleotidyltransferase. This may be a general feature in normal fetal development, since the differential processing of rearranged TCRD receptors found in the fetal gut has previously been identified in the human fetal thymus (27, 57). The mechanisms that generate AgR diversity appear, therefore, to operate differently for distinct TCRDV-DCRs.

In summary, our studies have shown that TCRDV-DCRs are expressed in the fetal liver and gut before thymus development and suggest that these tissues may be able to support the development of specific T cell populations. Further insights into the nature of the mechanisms that influence development in these sites will in large part be dependent upon the identification of the ligand(s) that are reactive with the receptors expressed by these cells.

	Footnotes

 
¹ This work was supported by grants to S.R.C. from the National Institutes of Health (AI-31972 and HL-51749) and the American Cancer Society (RPG-97-027). L.D.M. was supported in part by a National Institutes of Health Training grant (5-T32-CA-09140-20) and a Crohn’s and Colitis Foundation of America Fellowship.

² Current address: Department of Biochemistry, University of California at San Francisco, San Francisco, CA 94080.

³ Current address: Department of Pediatrics, Yale University, New Haven, CT 06510.

⁴ Address correspondence and reprint requests to Dr. Simon R. Carding, University of Pennsylvania School of Medicine, Department of Microbiology, Johnson Pavilion, Room 303A, Philadelphia, PA 19104-6076. E-mail address:

⁵ The nomenclature for human TCR- (TCRD) genes is as recommended in Reference 4. Rearranged TCRD genes are comprised of one of three variable (DV) gene segments (TCRDV101S1, TCRDV102S1A1T, and TCRDV103S1A1T), one, two or three diversity (DD) gene segments; one of four junctional (DJ) gene segments; and a single constant (DC) gene segment. The rearranged and expressed TCRDV-DD-DJ-DC receptor genes are designated in this paper as TCRDV1 or DV1R, TCRDV2 or DV2R, and TCRDV3 or DV3R, respectively.

⁶ Abbreviations used in this paper: IEL, intraepithelial lymphocyte; RAG; recombination activating gene; MNC, mononuclear cell; CDR, complementarity-determining region; N/P, new, template-independent or palindromic nucleotides.

Received for publication November 7, 1997. Accepted for publication February 18, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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