of May 18, 2018.This information is current as

During Fetal Development T Cell RepertoiresδγThe Generation of Human

Hayday and Simon R. CardingLaila D. McVay, Sheila S. Jaswal, Christine Kennedy, Adrian

http://www.jimmunol.org/content/160/12/58511998; 160:5851-5860; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/160/12/5851.full#ref-list-1

, 29 of which you can access for free at: cites 54 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Generation of Human gd T Cell Repertoires During FetalDevelopment1

Laila D. McVay,* Sheila S. Jaswal,2* Christine Kennedy,3* Adrian Hayday, † andSimon R. Carding4*

The nature of how humangd T cells are normally generated is not clear. We have used an RT-PCR assay and DNA sequencingto identify and compare d-encoded TCRs (TCRDs) that are generated de novo in the fetal gut, liver, and thymus and to determinewhen, where, and how the TCRD repertoire is established during normal embryonic development. RearrangedTCRDV genes arefirst expressed outside of the thymus in the liver and primitive gut between 6 and 9 wk gestation. Although DV1Rs and/or DV2Rspredominated, differences in the pattern ofTCRDV gene rearrangement and transcription in each tissue during ontogeny wereidentified. Specific, DV2-encoded TCRs are highly conserved throughout ontogeny in the tissues from the same and betweengenetically distinct donors. Although the thymic and intestinalgd T cell repertoires partially overlap early in development, theydiverge and become nonoverlapping during the second trimester, and the generation of the intestinalgd T cell repertoire ischaracterized by differences in the processing of DV1Rs and DV2Rs. Whereas the structural diversity of DV1Rs progressivelyincreases during gut development up to birth, DV2Rs have limited structural diversity throughout ontogeny. Together, ourfindings provide evidence for the ability of different fetal tissues to support the development ofgd T cells. The Journal ofImmunology,1998, 160: 5851–5860.

T he nature of how the humangd T cell repertoire is gen-erated is poorly understood. Unlikeab T cells, which arealmost exclusively dependent upon the thymus for their

development,gd T cells can be generated in extrathymic sites.Recently, we have shown that the fetal liver is an extrathymic sitefor gd T cell development during normal human development (1).CD31 lymphocytes that expressgd TCRs5 can be identified beforethymic development at 6 to 8 wk gestation. These cells expressTCRs that are essentially monomorphic and capable of respondingto microbial antigenic stimulation. Other studies that have identi-fied populations ofgd T cells unique to the liver are consistentwith our finding that the liver is a site ofgd T cell development (2,3). One question raised by these findings is whether other fetalhematopoietic tissues, such as the fetal intestine, are also sites of

humangd T cell development; if so, are the populations generateddistinct from those produced in the fetal liver and later in thethymus?

A comparative analysis ofTCRDVgene usage bygd T cells (4)in the fetal and adult gut has shown they are distinct. TCRDV21

T cells have been shown to predominate in the fetal gut (5),whereas TCRDV11 T cells have been shown to be the majorgd Tcell population in the adult human intestine, comprising#30% ofintestinal intraepithelial lymphocytes (IELs)6 (6–8). Interestingly,unlike the adult human thymus, the adult intestinal TCR-d andTCR-ab repertoires are oligoclonal (9–14). Recent studies suggestthat this oligoclonality is established between neonatal and adultlife (15). However, how this oligoclonality occurs is currently notknown.

The intestinal epithelia, similar to the thymic epithelium, may beable to support T cell development, and several lines of evidencesuggest a lymphopoietic capacity of the murine small intestine.Within the epithelial spaces, cells can be identified that expressmarkers that are characteristic of immature T cells, such as theCD32CD42CD81 cells which are TCR2 but contain abundantrecombination activating gene (RAG) transcripts (16). Uniquestructures in the crypts known as cryptopatches contain cells thatare phenotypically immature (17). Ectopically transplanted fetalintestinal grafts in athymic mice have been shown in some studies(18) to support T cells within the grafts and the generation of Tcells in the peripheral compartment. Positive and negative selec-tion of murine intestinal IELs (19–21) has been shown, demon-strating a capacity of the intestinal epithelium to provide a suitablemilieu for selection. Finally, the ability of intestinal epithelial celllines to promote the phenotypic maturation of T cells duringinvitro culture suggests that the intestinal epithelia may be able toinduce or support the differentiation of T cell progenitors (22–24).

Despite these studies, the possibility that the human fetal intes-tine can support T cell development has not been addressed. To

*Department of Microbiology, University of Pennsylvania School of Medicine, Phil-adelphia, PA 19104; and†Department of Biology, Yale University, New Haven, CT06520

Received for publication November 7, 1997. Accepted for publication February18, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.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 Fran-cisco, 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 ofPennsylvania School of Medicine, Department of Microbiology, Johnson Pavilion,Room 303A, Philadelphia, PA 19104-6076. E-mail address: carding@mail.med.upenn.edu5 The nomenclature for human TCR-d (TCRD) genes is as recommended in Reference4. Rearranged TCRD genes are comprised of one of three variable (DV) gene seg-ments (TCRDV101S1,TCRDV102S1A1T, andTCRDV103S1A1T), one, two or threediversity (DD) gene segments; one of four junctional (DJ) gene segments; and asingle constant (DC) gene segment. The rearranged and expressed TCRDV-DD-DJ-DC receptor genes are designated in this paper as TCRDV1 or DV1R, TCRDV2or DV2R, and TCRDV3 or DV3R, respectively.

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

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

understand more clearly how thegd T cell repertoire is generatedat the molecular level and the nature of the mechanisms that mightcontribute to their selection and survival, we have conducted adetailed ontogenetic analysis ofgd TCR gene expression in theintestine, comparing it with that seen in the liver and thymus dur-ing human fetal development. We have used a large number ofsamples from various ages during fetal development to analyze theexpression ofTCRDVgenes in the fetal liver and gut before thymiccolonization; this expression was also analyzed in the thymus,liver, and gut after thymic colonization from the same donors whenpossible. We provide evidence for the ability of different fetal tis-sues to influence the development of humangd T cells and identifyfeatures of the TCRDV intestinal repertoire that distinguish it fromthose expressed in other fetal tissues.

Materials and MethodsTissue samples and cell isolation

Fetal tissue samples were procured by the Anatomic Gift Foundation (Lau-rel, MD) with the approval of the University of Pennsylvania Committeefor Studies Involving Human Subjects (Philadelphia, PA). The gestationalage of individual samples was determined according to the Carnegie em-bryonic staging system (25, 26). Where possible, microscopic examinationwas also used to establish the absence or presence of a thymic rudiment.The liver, thymi, and intestine samples obtained from same donors andused 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 inthis study is shown in Table I.

RT-PCR analysis of the TCRVd 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 pre-cipitation. We used;10mg of total cellular RNA to synthesize cDNA withrandom hexanucleotides. The amount of cDNA synthesized from eachRNA sample was normalized according to the amount of humanb-tubulinPCR product generated from 1ml of cDNA that had been amplified usingb-tubulin-specific primers (28). The primers and the RT-PCR assay usedfor theTCRDVgene segment analysis have been described previously (27).Briefly, oligonucleotide primers specific forDV1, DV2, or DV3in combi-nation with aDC gene segment primer were used to amplify cDNA in a 10ml reaction volume using an air-driven thermal cycler (Idaho Technology,Idaho Falls, ID). A nested PCR assay was used to analyze theDV-DJusage(1). The products of DV-DJ amplification were electrophoresed, andTCRD PCR products were gel purified and quantitated by DNA fluorom-etry. Equivalent amounts of each cDNA were used as a template for asecond amplification using the same forward DV primer and a reverseprimer that was specific for each of the four humanDJ genes. The reverseprimers corresponding to theDJ gene segments used for the analysis ofDJgene usage among amplified DV1-DCRs, DV2-DCRs, and DV3-DCRshave been described previously (1). ForDV/DJ gene analysis, DV1, DV2,and DV3 oligos that lie at the 39 end of eachDV gene and internal to thepriming oligos were used as probes. Individual DV1-DJ1–4, DV2-DJ1–4,and DV3-DJ1–4R cDNA clones were used to determine the optimum con-ditions for the proportional amplification ofDV/DJ genes withoutcross-priming.

mAbs and flow cytometry

The murine mAbs reactive with human TCR-gd and CD8a proteins werepurchased from T Cell Diagnostics (Boston, MA). The anti-human CD3Ab was obtained from Becton Dickinson (Sunnyvale, CA). The anti-humanab Ab, BMA031, was obtained from Behringwerke AG (Marburg, Ger-many). Before staining, fetal liver and gut mononuclear cell (MNC) prep-arations were depleted of erythroid cells by immunomagnetic depletion(28) using an anti-human glycophorin A-specific Ab (Immunotech, West-brook, ME); the remaining 10 to 15% of cells were incubated with Abs tohuman FcRg II (clone 4.3, American Type Culture Collection (ATCC),Manassas, VA) and FcRg III (clone 3G8, ATCC) to block the nonspecificbinding of fluorochrome-conjugated Abs. For the two-color flow cytomet-ric analysis of fetalgd T cells, between 23 105 and 13 106 cells wereinitially stained with biotinylated primary Abs and then stained with phy-coerythrin-conjugated Abs and streptavidin-Red613 (Life Technologies,

Gaithersburg, MD) as described previously (29). Stained cells were run ona FACScan (Becton Dickinson) and analyzed using Lysis II software.

DNA sequencing

PCR-amplified DV1-DCR and DV2-DCR cDNAs were agarose-gel puri-fied (Glas Pac/GS, National Supply Company, San Rafael, CA) and cloned

Table I. Postthymic fetal tissue samplesa

Age (wk)

Code No.

Thymus Liver Intestine

9 *9-32043T 9-32043L 9-32043G*9-14330T 9-14330L 9-14330G*9-14449T 9-14449L 9-14449G9-32064T 9-32191L 9-26017G9-32067T 9-14447L 9-26023G

10 *10-31391T 10-31391L 10-31391G10-14429L10-27485L

11 11-14322T 11-27397L 11-14227G12 12-14422L 12-0039G13 *13-22156T 13-22156L 13-22156G

* 13-22452L 13-22452G13-19325L

14 *14-19322T 14-19322L 14-19322G*14-22541T 14-22541L 14-22541G*14-20851T 14-20851L* 14-003918L 14-003918G

14-222543G15 *15-19504T 15-19504L 15-19504G

*15-19467T 15-19467L 15-19467G*15-22199T 15-22199L*15-19019T 15-19019G* 15-03920L 15-03920G* 15-22378L 15-22378G* 15-19215L 15-19215G

15-19273L 15-89G15-20475L 15-83G15-22480L 15-22392G

15-22528G15.5 *15.5-19501T 15.5-19501L 15.5-19501G

*15.5-185T 15.5-185L 15.5-185G15.5-19469L 15.5-19469G

15.5-531G15.5-19027G

16 *16-22248T 16-22248L 16-22248G*16-222256T 16-22256L 16-22256G*16-22157T 16-22157G*16-22395T 16-22395G* 16-22180L 16-22180G* 16-22353L 16-22353G* 16-22446L 16-22446G* 16-19222L 16-19222G

16-2890L 16-222445G16-003902L

17 *17-20923T 17-20923L 17-20923G17-190227T 17-10588L 17-6967G

17-20804G18 *18-18wT 18-18wSmInt-G

18-18wLgInt-G18-19228T 18-19461L18-19918T18-22182T

22 22-22wT 22-01L23 23-19913T

23-19919T33 *33-01T 33-01L 33-01G

Total 31 42 44 5 117

a The identity of the tissue source is indicated by -T, -L, or -G for thymus, liver,or gut (intestine). Tissues from the same donor are identified by the same number andan asterisk (*).

5852 HUMAN gd T CELL DEVELOPMENT

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

into the pGEM-T vector (Promega, Madison, WI) or the TA vector (In-Vitrogen, San Diego, CA). Plasmid minipreps from randomly picked col-onies were used to obtain DNA for the sequencing of both strands by thedideoxynucleotide chain termination method using modified T7 polymer-ase (Sequenase Version 2.0, U.S. Biochemical, Cleveland, OH).

ResultsAn 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 theembryo. The primitive gut is divided into the foregut, the midgut,and the hindgut, which can be distinguished on the basis of arterialblood supply; the celiac, superior mesenteric, and inferior mesen-teric arteries vascularize the foregut, the midgut, and the hindgut,respectively. Branches of the celiac artery vascularize outgrowthsof the foregut, in particular the liver, early during wk 4, establish-ing a common blood supply and a link between the developing gutand liver (30). This finding has important implications in under-standing the origin of the first lymphoid cells. Intestinal IELs havebeen identified at 11 wk (31), and lymphocytes in the lamina pro-pria have been identified at 14 wk (32). Peyer’s patch aggregatesare first evident at 11 wk; these aggregates are diffusely colonizedwith T and B lymphocytes at 16 wk and with defined zones of thegerminal follicle of Peyer’s patches at 19 wk (33), suggesting thatthe anatomic organization of the fetal intestine is apparent duringthe first trimester.

Study outline and samples

Our study was designed to address how the humangd T cell rep-ertoire is normally generated in vivo during fetal development.Specifically, we addressed when and in which tissuesTCRDV-DCgenes are first activated, whether the fetal gut is a site ofgd T celldevelopment, and, if so, whether the populations generated aredistinct from those found in other sites of T cell development, suchas the fetal liver and thymus.

We focused on the analysis ofgd T cell development in the fetalintestine, liver, and thymus before and after thymic developmentand colonization by T cell precursors. TCR gene expression wasrestricted to the analysis of the TCRDV repertoire since, in con-trast to TCRGV mRNA expression (34), TCRDV mRNA expres-sion correlates well with protein expression. Although sixDVgenes have been described (35), subsequent analyses have shownthat TCRDV4, TCRDV5, and TCRDV6 are TCRA genes(TCRAV6.2,TCRAV21, andTCRAV17, respectively) (35–38).Since rearrangedTCRDV1-,TCRDV2-, andTCRDV3-DCgenespreferentially rearrange toDJ gene segments to form TCRDRsrather than toAJ gene segments to form TCRARs, our study wasrestricted to these three predominantTCRDV-DJ-DCgenes.

Since the quantities of fetal tissues obtained were small, and cellnumbers were low, it was not possible to routinely perform anal-yses on isolated TCR1 cells. Instead, an RT-PCR assay was usedto characterize TCRDVR transcription. The specificity of the prim-ers used was verified using individual TCRDV-DC cDNAs clonedfrom gd T cell lines or clones. No PCR product was amplified inthe absence of cDNA or when an irrelevant cDNA (fetal kidney)was used as a substrate. Over a concentration range of 104-fold, theamount of PCR product obtained was proportional to the amountof substrate cDNA as detected by Southern blot hybridization. Thisfinding was determined by the amplification of a range of concen-trations of T cell cDNA diluted into fibroblast cDNA as describedpreviously (28). This assay cannot, however, provide any quanti-tative data concerningTCRDVgene expression, and the conclu-sions reached in this study rely on detecting changes or differencesin the relative levels ofTCRDVgene expression.

To identify and analyze TCRDVR transcription that may occurindependently of any thymic influence, liver and gut samples wereobtained before thymic colonization at 6 wk gestation. In addition,thorax/thymus, liver, and gut samples were obtained before or co-incident with thymic colonization at 8 wk gestation. In some cases,it was possible to obtain multiple tissues from the same donor. Toovercome the potential variability of human tissue and to analyzeTCRDVgene rearrangement at later times during fetal develop-ment,.100 additional tissue samples were used (Table I).

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

Our first indication that TCRDVR transcripts might be activated inthe fetal liver and gut independently of the thymus was detectingexpression of RAG-1 and RAG-2, which are normally required forthe rearrangement ofTCRgenes (39, 40), in the 6 wk liver and gut(Ref. 1 and data not shown). Consequently, fetal liver MNCs andgut tissue mRNA samples between 6 and 9 wk gestation wereanalyzed for evidence of TCRDV-DJ using a sensitive RT-PCRtechnique that had been previously optimized for the analysis ofTCRGVand TCRDVgenes in the human fetal thymus (27) andliver (1), as described inMaterials and Methods.

Although the profile of TCRDVR transcripts in the liver andgut were very similar at 6 wk, there were differences in theextent of DJ usage in TCRDV1-, TCRDV2-, and TCRDV3-encoded receptors (Fig. 1). Whereas TCRDV1R (Fig. 1A) andTCRDV2R transcripts (Fig. 1B) almost exclusively usedDJ1and/or DJ3 genes, all of theDJ gene segments were used inDV3R transcripts (Fig. 1C). In the 8 wk samples, it was pos-sible to identify profiles of TCRDV-DJR transcripts that wereunique to each tissue. In the gut, the restricted use ofDJ1 andDJ3 in TCRDV1-encoded receptors (Fig. 1A) and the use of allDJ gene segments in TCRDV3R transcripts (Fig. 1C) distin-guished it from the profiles seen in the thymus and liver. Inaddition, the predominance of DV3-DJ3R transcripts in the 8wk liver distinguished it from the thymus, in which TCRDV3R-transcripts were barely detectable (Fig. 1C).

Although there was some variability in the profile ofTCRDV-DJ transcripts in the 9 wk tissue samples obtained fromdifferent donors, several interesting features of TCRDV-DJ tran-scription were identified by comparing the profiles obtained fromtissues of the same donor. The profile of TCRDV1-DJ transcriptsin the gut was strikingly similar to that seen in the thymus (Fig.1A). In addition, the TCRDV2-DJ transcripts detected in the gut,thymus, and liver were also very similar (Fig. 1B). In contrast, theprofiles of TCRDV3-DJ transcripts were more variable in tissuesof the same donor (Fig. 1C). In summary, these results show thatall TCRDVgenes are expressed in the liver and gut before thymicdevelopment, and that the pattern of TCRDV-DJ transcripts wasdifferent for each of theTCRDVgenes.

To extend this analysis and determine whether and how theTCRDV repertoire changes later in fetal development, an addi-tional 117 fetal tissue mRNA samples obtained from donors be-tween 9 and 33 wk gestation (Table I) were analyzed. The South-ern blot analysis of PCR-amplifiedTCRDV-DCgenes from mostof these fetal tissues is summarized in Table II.

TCRDV transcription in human fetal tissues throughoutontogeny

The pattern of TCRDVR transcripts changes in conjunction withthymic colonization (Table II). TCRDV1 predominated between15 and 16 wk, while the expression of the otherTCRDVgenes wasonly detected after a long (4-day) exposure. TCRDV1 andTCRDV2 were more strongly expressed than TCRDV3 transcripts

5853The Journal of Immunology

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

between 17 and 22 wk and at 33 wk gestation, with TCRDV2being predominant. Within the postnatal thymus, DV1 expressionwas predominant, and DV2 expression could not be detected, aswe (1) and others (41–44) have previously observed.

The pattern of TCRDVR transcripts also changed during fetalliver ontogeny and was distinct from that seen in the thymus (Ta-ble II). Whereas most DV transcripts were detected between 9 and14 wk in the thymus, the profile of transcripts in the liver was morerestricted, with DV2 predominating in the majority of samplesanalyzed. Indeed, DV2 was the only transcript detected in five ofsix 16 wk samples. At 17 to 22 wk, all DV transcripts were de-tected, with DV2 remaining predominant.

In the fetal intestine, most TCRDVR transcripts were expressedthroughout 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 waspredominantly seen at 9 to 11 wk, 15 to 15.5 wk, and 33 wk. Theapparent increase in the amount of DV2 transcripts detected asontogeny proceeds (Table II and data not shown) may reflect anincrease in the number of TCR-d1 cells or in the number of tran-scripts per cell. This increase is coincident with the time in gutdevelopment during which it is possible to isolate sufficient num-bers ofgd 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 betweenage-matched samples, consensus patterns of expression were ap-parent (Table II). For example, one striking feature was the sin-gular predominance of DV1R transcripts in the 15 to 16 wk fetalthymus and DV2R transcripts in the 16 wk fetal liver. Also, theoverall pattern of DV3 transcripts was less variable than DV1 orDV2 transcripts in all of the samples examined. DV1 was predom-inant in both the 3-yr postnatal thymus and the adult intestine, aspreviously described (9, 10, 27, 41–43) and is included here todemonstrate the comparison of TCRDVR transcripts in fetal andadult tissues.

Structure of TCRDV1R transcripts expressed before and afterthymic colonization

Although the normal adult intestine is characterized by the oligo-clonality of the TCR-d repertoire (9–14) that becomes apparentafter birth at between 14 and 17 years of age (15), the analysis ofoligoclonality in fetal development had only been studied at a sin-gle timepoint (20 wk) (15). Thus, we were interested in determin-ing 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 gestationalages was analyzed. Since DV1 was the predominantDV gene ex-pressed in the fetal thymus after colonization as well as in thepostnatal thymus and the adult intestine (9, 10, 27, 41–43), thisreceptor was initially chosen for DNA sequence analysis. In ad-dition, since we have shown here that DV2R transcripts can bedetected as early as 6 wk in the primitive gut (Fig. 1B), this re-ceptor was also chosen for DNA sequence analysis.

Table III is a compilation of DV1-DJ and DV2-DJ DNA se-quences 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 inthese very early samples (Table IIIA) with a single DV-DC PCRamplification, the PCR products were gel-purified, quantitated, andreamplified in a nested PCR using DV and DC oligos that werelying internal to those used for the initial amplification reaction.Despite this additional amplification step, it was only possible toisolate 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-gd mRNA-expressing cells in these early fetal tissues.In addition, we were unable to obtain any DV cDNA clones from

FIGURE 1. TCRDV-DJgene expression in fetal hematopoietic tissuesbefore (6–8 wk) and immediately after (9 wk) thymic colonization. cDNAsamples from fetal liver and thymic MNCs or primitive gut tissue mRNAwere amplified using DV1 (A), DV2 (B), or DV3 (C) and DC-specificprimers. Aliquots of the DV-DC PCR products were then reamplified bynested PCR using the same DV primer and a reverse primer that wascomplimentary to each of the knownDJ genes. Amplified DV-DJ PCRproducts were detected by Southern blot analysis using a DV-specificprimer that was internal to the DV PCR primer as a probe. Control PCRsamples contained either no cDNA (A–C) or fetal kidney cDNA (A). Au-toradiography lasted for 1 day. The first digit of the sample code numberrepresents the gestational age in weeks, and the hyphenated number rep-resents the code number used to identify each sample.

5854 HUMAN gd T CELL DEVELOPMENT

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

the 6 wk fetal liver sample. In the 6 wk gut, two distinct receptorswere identified; they comprised one or moreDD gene segmentsand primarily used aDJ1 gene segment. These features are un-characteristic of “fetal” receptors that usually have a singleTCRDDgene segment and limited junctional diversity. Neither ofthese receptors were found in the fetal intestine, thymus, or liver atlater stages of gestation.

The number of structurally distinct DV2Rs identified was small,with specific receptors being present in more than one tissue of thesame donor and in tissues of different donors (Table IIIB). Tworeceptor sequences represented at least 50% of those found in thethymus, liver, and gut from the 6 and 8.7 wk donors. For example,in the 8.7 wk liver, 21 of 23 productive sequences were identical;this receptor sequence was the most highly represented sequencein the thymus of this donor, in the liver of the 6 wk donor, and inliver samples obtained later in gestation (15). In contrast to DV1-encoded receptors, the structure of DV2-encoded receptors moreclosely resembled that of fetal receptors. A single DD segment wasused, and N/P nucleotide insertion was minimal (Table IIIB). Inaddition, three of fourDJ gene segments were used for DV2-encoded receptors, althoughDJ1 andDJ3 predominated. The ma-jority of DV2-DJ1 (30 of 32) and DV2-DJ3 (44 of 50) receptorswere in frame, and the predominance ofDJ1 and DJ3 was con-sistent with the mRNA expression profile as detected by RT-PCR(Fig. 1). In contrast, DV2 sequences in the thymus were diverse.Although 17 of 22 receptor sequences used DJ3, 18 of 22 se-quences were productive, and 7 of 18 were represented only once.The remaining four productive DV2-DJ3 thymic sequences werethe same as those found in the fetal liver of the same donor.

A comparison of the predicted amino acid sequence forTCRDV1- and TCRDV2-encoded receptors clearly shows that thecomplementarity-determining region (CDR)3 of the DV1Rs is

longer than the DV2Rs (Table IV), further illustrating the differ-ence between DV1R and DV2R transcripts in 6 to 9 wk fetaltissues.

The structure of fetal gut TCRDV1Rs between the first and thirdtrimester of gestation

We chose to analyze DNA sequences in 15 wk intestinal samples,since our analysis of TCRDV mRNA expression (Table II) iden-tified a striking change inTCRDVgene expression, particularly inthe intestine at this time point, and sufficient numbers of cells forphenotypic analysis could be isolated. At this point in develop-ment, the structural diversity of DV1Rs expressed in the intestinewas comparable with those expressed earlier during ontogeny (Ta-ble IVA). Of the 15 DNA sequences obtained, 10 were productiveand were represented only once or twice (Table IVA). This diver-sity in receptor sequences was attributable to N/P nucleotide in-sertion and exonucleolytic nibbling, particularly at the 59 and 39ends of theDD3 gene segment (data not shown). The CDR3lengths of these receptors were comparable with those expressed inthe 6 and 8 wk intestine (Table IVA). Thus, there is no evidenceof TCRDV1 oligoclonality in the fetal intestine at this time point.

A DNA sequence analysis of 24 DV1Rs from the 33 wk gutsample demonstrated that the diversity of this receptor reper-toire was increased further. Of the 24 sequences obtained, nonewas represented more than once, and they resembled the struc-ture of neonatal/adult receptors, with multipleDD gene seg-ment usage and N/P nucleotide insertions present (data notshown). A total of 13 of 24 DV1Rs were in frame (Table IVA)and further illustrate the increase in the CDR3 length of thesereceptors at this time.

Table II. Summary of TCRDV-DC transcription during ontogenya

Fetal Thymus Fetal Liver Fetal Intestine

Age(wk) Vd1 Vd2 Vd3

Age(wk) Vd1 Vd2 Vd3

Age(wk) Vd1 Vd2 Vd3

9 11 11 11 9 1 11 1 9 11 1 19 11 11 1 9 11 11 1 9 11 11 1

10 11 11 1 10 1 11 1 10 1 11 111 11 11 1 11 11 11 1 11 11 11 111 11 11 11 11 1 11 1 11 1 11 112 1 12 1 11 1 12 1 11 113 1 1 1 13 1 11 1 13 1 11 114 1 1 1 13 1 11 14 1 11 114 11 1 1 14 1 11 1 14 11 11 115 11 1 14 1 11 15 1 11 115 11 1 15 11 11 15 11 11 115 11 15 11 15 11 11 115 11 15 11 1 15.5 11 11 115.5 11 15.5 11 1 1 15.5 1 11 115.5 11 15.5 1 11 15.5 1 11 116 11 16 11 16 1 11 116 11 16 11 16 1 11 116 11 16 11 16 1 11 116 11 11 1 16 11 16 1 11 117 11 11 1 16 11 16 1 11 118 11 11 1 16 1 17 1 11 118 11 11 1 16 1 11 1 18 1 11 122 11 11 1 17 1 11 18 1 11 133 11 11 1 17 1 11 1 33 11 11 1

18 1 11 122 1 11 1

3 yr 11 11 42 yr 11 1 1

a 11 denotes strong TCRDR transcription and1 denotes relatively weaker TCRDR transcription. Blank spaces denote thatno TCRDR transcription was detected. The samples at the bottom of the table denote postnatal and adult time points.

5855The Journal of Immunology

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Table III. DNA sequence analysis of TCRDRs from 6 to 9 wk liver, gut, and thorax/thymus of the same donora

A. TCRDV1-DD-DJ-DCRs No. seqs.

DV1 DD1 DD2 DD3 Code no.: 6-01 8.7-691 9-14330G E

GGG GAA CT N/P GAAATAGT N/P CCTTCCTAC N/P ACTGGGGGATACG N/P DJ G T G T L G

GGG AC GGGGG TGAGGGGACTCCAGGAC ACC GAT(1) (1) 8 16GGG GAA C T ACTGGGGGAT TTG ACA (2) (1) 3GGG GAA C TAGGCC GGGAT CCCTTAAAAGGGG CC GAT(1) (1) 6GGG GAA C GGTCAGGGGCCCT CTTC GT ACTGGGGGAT TCGGTGT AC ACC GAT(1) (1) 12 12 16GGG GAA G CCTTCC GACCATC ACG CACCTT C ACC GAT(1) (1) 1

7 11 16 12 12 16

B. TCRDV2-DD-DJ-DCRs No. seqs.

DV2 DD1 DD2 DD3 Code no.: 6-01 8.7-691C D T

TGT GAC ACC N/P GAAATAGT N/P CCTTCCTAC N/P ACTGGGGGATACG N/P DJ L G T L G

TGT GAC ACTGGGGGATAC ACC GAT(1) (1) 3TGT GAC GT ACTGGGGG AC ACC GAT(1) (1) 2 3 2 18TGT GAC AC ACTGGGGGAT ACC GAT(1) (1) 1TGT GAC ACC G CTGGGGGATAC ACC GAT(1) (1) 1TGT GAC ACC TT ACTGGGGG CTTCC AC ACC GAT(1) (2) 1TGT GAC ACC TGGGGGATAC C GAT (1) (2) 1

4 5 2 21TGT GAC ACC TGGGGGATACG C TCC TGG(3) (1) 8 4 21 1TGT GAC GATAT GGGATAC C TGG (3) (2) 1 1TGT GAC A T ACTGGGGGATACG C TCC TGG(3) (2) 1TGT GAC A T ACTGGGGGAT CCC C TCC TGG(3) (2) 1TGT GAC ACC TGGGGGATAC CCGT C TGG (3) (1) 1TGT GAC ACC G CTGGGGGATAC TCC TGG(3) (1) 1TGT GAC AC GGGTATT ACTGG C TCC TGG(3) (1) 1TGT GAC ACC GGGGG TCTTAAG C TCC TGG(3) (2) 1TGT GAC AC ACT CTGGGGGATA G C TCC TGG(3) (2) 1TGT GAC ACC TGGGG TAG C TCC TGG(3) (1) 1TGT GAC ACC TGG (3) (1) 2TGT GAC ACC TGGGGGATA GCA (2) (1) 2TGT GAC ACCTGT GAC ACC GGGGGATAC TTG ACA (2) (1) 1TGT GAC GT ACTGGGGGATAC A GCA (2) (1) 1TGT GAC A AGG CTGGGGGATACG CGAG CTC (2) (2) 1

10 5 22 23 23

a In-frame joints are labeled1, while out-of-frame joints are labeled2. The identity of the DJ gene segment is indicated by the number 1, 2, 3, or 4. No. seqs. is the frequency with which a particular sequence was found among cDNA clones.

5856H

UM

AN

gd

TC

ELL

DE

VE

LOP

ME

NT

by guest on May 18, 2018 http://www.jimmunol.org/ Downloaded from

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

To determine whether the structural complexity of DV2R geneswas similar to DV1R genes and whether the oligoclonality of theDV2 repertoire was apparent at all during fetal life, we analyzedthe 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 usedfor the analysis of DV1Rs.

Of the DV2 cDNAs sequenced from the 6 and 8.7 wk gut, thesame sequence and receptor was predominant in each sample (Ta-ble IVB). Of the DV2 cDNAs sequenced from the 15 wk gut, 12

of 16 were productive, of which two represented approximatelyhalf (6 of 12) of those sequenced. Compared with DV1Rs, theDV2Rs were structurally simpler, with limited N/P nucleotide in-sertion and exonucleolytic processing and use of a singleDD genesegment. The CDR3 lengths of 33 wk DV2Rs (Table IVB) werestrikingly similar to those identified in the 15 wk sample. In ad-dition, while there is a predominance of DV2Rs using the DJ1gene segment in the first trimester, this changes to a predominanceof DJ3 use in the third trimester. Thus, while DV1Rs between 6and 33 wk were extensively modified and structurally diverse,DV2Rs in the same donors were fetal in structure.

Thymic and intestinalgd T cells are phenotypically distinct anduse structurally distinct, nonoverlapping TCRDV2Rs

Since the expression ofgd TCR and associated differentiation Ags(CD2, CD3, CD4, and CD8) may distinguish between populationsof fetal gd T cells, a phenotypic analysis of intestinal and thymicgd T cells that had been isolated from a single 15 wk donor wasperformed. This analysis could not be performed on earlier sam-ples, because it was not possible to isolate sufficient numbers ofcells. Thymocytes and gut lymphocytes isolated from the same 15wk donor were stained using Abs directed againstgd TCR, CD3,and CD8a cell surface glycoproteins (Fig. 2).gd T cells comprised2% of CD31 thymocytes and were CD82 (and CD42, data notshown). 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 my-elomonocytic and B cell lineage markers CD11b and CD19, re-spectively (data not shown). Of these cells, 8 to 9% were CD31,of which virtually all cells weregd1 (Fig. 2). In contrast to TCR-gd1 thymocytes, intestinalgd T cells were uniformly CD8a1.Thus,gd cells comprise the vast majority of those T cells in the 15wk gut that can be distinguished from the thymic population on thebasis of CD8a expression. Our data does not, however, enable usto establish when CD8a expression is acquired by intestinal T cellsand whether it is restricted to cells residing in the intestinal epi-thelium or is acquired by CD8 thymic migrants that populate theintestine.

To determine whether 15 wk intestinal and thymicgd T cellsexpressed distinct receptors, a comparative analysis ofTCRDV2Rs was undertaken using fetal thymus and gut samplesfrom the same donor (No. 15-185T/15-185G). While all (8 of 8) ofthe receptors in the fetal thymus were distinct,.50% (6 of 11) ofthe sequences in the fetal gut sample were represented by twosequences (Table V). Of note, there were no overlapping se-quences detected in the thymic and gut samples analyzed.

DiscussionSeveral findings emerge from this work. First, in agreement withour previous study (1), rearrangedTCRDV-DCgenes are initiallyexpressed outside of the thymus and are expressed in the liver andprimitive gut between 6 and 9 wk gestation. Second, the patternsof TCRDVR transcripts change in the tissues examined duringontogeny, which are characterized by a predominance of DV1Rand/or DV2R. Third, DV2-encoded TCRs are highly conservedthroughout ontogeny in the tissues from the same and geneticallydistinct donors. Fourth, although the thymic and intestinal DV2Rrepertoires partially overlap early in development, they divergeand become nonoverlapping during the second trimester. Fifth, thegeneration of the intestinalgd T cell repertoire is characterized bydifferences in the processing of DV1R and DV2R genes. Whereasthe structural diversity of DV1Rs is apparent in ontogeny and pro-gressively increases during gut development, DV2Rs have limited

Table IV. Amino acid sequence of TCRDRs from fetal intestinea

A. TCRDV1Rs DV1 ND1ND2ND3N DJ No. Seqs.

6 wk (No. 6-01)GE LGRDPLKGA D (1) 6GE AFRPSRTF TD (1) 1

8.7 wk (No. 8.7-691)G TGVRGLQD TD (1) 16

9 wk GE RSGALFVLGDSVY TD(1) 16

15 wk (No. 15-185G)GE RGY TD (1) 1G VLGD TD (1) 1GE LGDT L (2) 1GE TGGA P (3) 1GE RGYG PL (4) 1GE YTGGY TD (1) 1GE LRGLPYS TD (1) 1GE AIRPSRTF TD (1) 2G TGGEGTPD TD (1) 1

33 wk (No. 33-01G)GE LWGY TD (1) 1G LSWP LTA (2) 1G YWGC SWD(3) 1G PPAFN TD (1) 1GE PTKGH TD (1) 1GE RSRGLH TD (1) 1G AGQGGP SWD(3) 1G ILHSCPH TD (1) 1G VGFLRHWG D (1) 1GE LARLGDTGY TD (1) 1G DPFDSVYWGKA DD(3) 1GE LVESPYWGIEGQV TD(1) 1G DGFLQVLGDTRGD LTA(2) 1

B. TCRDV2Rs DV2 ND3N DJ No. Seqs.

6 wk (No. 6-01)CD VLGD TD (1) 2CDT AGGY TD (1) 1

8.7 wk (No. 8.7-691)CD TGGY TD (1) 3CD VLGD TD (1) 18

15 wk (No. 15-185G)CDT WG TD (1) 1CDT GGY TD (1) 4CDT LGD TD (1) 1CD WGR SWD(3) 2CDT WGIR SWD(3) 1CD KLGDT A (2) 1CDT MTGGY SWD(3) 1CD VLGDT SWD(3) 1

33 wk (No. 33-01G)C VV SWD (3) 1CD EA WD (3) 1CD WGS SWD(3) 1CD GILG WD (3) 1CDT GLGDTF SWD(3) 1

a The identify of the DJ gene segment is indicated by 1, 2, 3, or 4. No. Seqs. is thefrequency with which a particular sequence was found among cDNA clones.

5857The Journal of Immunology

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

structural diversity throughout ontogeny. Finally, our results areconsistent with the recent findings that the oligoclonality of theintestinal DV1R repertoire is established after birth (15).

The expression of RAG mRNA and TCRDVR transcripts in thefetal gut before thymic organ formation and colonization and bylymphocyte progenitors is consistent with the observation that thegut, like the fetal liver (1), is an extrathymic site of de novo generearrangement. However, the exact sequence of TCRD gene rear-rangment and expression, the site(s) in which it is initiated, and the

mechanism by which the gut might be seeded with progenitors arenot known. Although we can exclude the thymus as the site oforigin of cells expressing the TCRDVR transcripts that are presentin the 6 wk primitive gut, our studies cannot discriminate betweenthe following possibilities: 1) that these cells originated in the liverand traveled to the gut, 2) that they were generated in the gut andseeded to the liver, or 3) that they were generated independently ineach tissue. The identification ofgd and IEL progenitor popula-tions in the human intestine would help resolve this issue. Al-though putative IEL progenitor cell populations have been identi-fied within distinct clusters of cells or cryptopatches (17) in themurine intestinal mucosa, analogous populations of cells have notbeen found in the human intestine to date.

The striking diversity of TCRDV1Rs expressed in the 6 wkprimitive gut distinguishes thegdR expression in this site from thatseen in either the thymus or liver at any time during fetal devel-opment. The structure of these receptors resembles that seen inadults, suggesting that they may be of maternal origin. The inabil-ity to detect terminal deoxynucleotidyltransferase activity in fetaltissues until 12 wk gestation (45) and the presence of Vd11 gd Tcells within the placental decidua at this time during fetal devel-opment (46) is consistent with this possibility. However, if DV1-expressinggd T cells can cross the placental barrier and gain ac-cess to the fetal circulation, this action would be a property that isunique to this subset ofgd T cells, since we have been unable toobtain any evidence for the presence of maternally derived DV2-expressinggd T cells in fetal tissues (1).

Distinctive patterns of TCRDVR transcripts and receptor struc-ture in different tissues of the same donor may reflect the traffick-ing of different DV1 populations to and from distinct anatomicalsites at different times during development. The establishment of acommon blood supply between the liver and intestine after wk 4 ofgestation and the vascularization of the thymus after 8 wk gesta-tion (30) provides a means by which cells generated in the liver or

FIGURE 2. Human fetal thymic and intestinalgd T cells are phenotypically distinct. Thymocytes and intestinal MNCs from a single 15-wk donor werestained with anti-CD3/TCR and CD8a-specific Abs and analyzed by flow cytometry as described inMaterials and Methods. The level of staining withfluorochrome-labeled, isotype-matched Abs of irrelevant specificity was used to determine the frequency of the positive cells shown in each quadrant ofthe contour plots.

Table V. Amino acid sequence of TCRDV2Rs from fetal thymus andintestine of the same donor (No. 15-185)a

Thymus

TCRDV2-DD-DJ Sequences

DV2 N-DD3-N DJ No. Seqs.

ACDT GGY K (1) 1ACDT GA LTA (2) 1ACDT GGY SWD (3) 1AC GGY WD (3) 1ACDT LGS SWD (3) 1ACDT VLGDI SWD (3) 1ACDT VLGDS SWD (3) 1ACD NVLGDTS SWD (3) 1

Intestine

TCRDV2-DD-DJ Sequences

DV2 N-DD3-N DJ No. Seqs.

ACDT WG TDK (1) 1ACDT GGY TDK (1) 4ACDT MTGGY TDK (1) 1ACD KLGDT A (2) 1ACD WGT SWD (3) 2ACDT WGIR SWD (3) 1ACD VLGDT SWD (3) 1

a The identity of the Jd segment is indicated by 1, 2, 3, or 4. No. Seqs. is thefrequency with which a particular sequence was found among cDNA clones.

5858 HUMAN gd T CELL DEVELOPMENT

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

gut can transit between each tissue and to the thymus. The expres-sion of adhesion molecules bygd populations or their progenitorspromoting their adherence to, and retention by, various stromalcell populations provides a possible mechanism for the distributionand retention of specificgd cells in different tissues. Indeed, thepreferential localization of Vd11 gd cells to intestinal epithelia isassociated with the expression of a particular integrin, E-cadherin,(47). In addition, we and others have also shown that among hu-mangd T cells, Vd11 cells can preferentially adhere to fibroblastsnormally found in the skin (48) and colon (49). An alternative butnot mutually exclusive explanation for these differences in tissueTCRDV profiles is that they may be a consequence of the selectivesurvival, expansion, or death of particular Vd-expressing cells byvarious factors (e.g., Ags and cytokines) that are present in themicroenvironments of each tissue.

The conservation of identical, particularly DV2, receptors is an-other striking feature of humangd T cell development. In additionto the DV2Rs first detected in the 6 wk fetal liver and gut, recep-tors with the same sequences can be found among a panel ofgd Tcell clones derived from the 14 wk fetal liver (1) and among cDNAclones from the thymus and peripheral blood of adults (50). Al-though the nature of the mechanisms responsible for this bias inreceptor usage are not known, it seems likely that both intrinsic(genetic) or extrinsic (environmental) factors are involved. Ananalysis of rearrangedDV/DC genes on both chromosomes of hu-man peripheral blood, thymus, and intestinalgd T cell clones hassuggested that preferential gene rearrangement accounts for thebias in TCRV region usage in humangd T cells (51). However, weare unable to exclude the possibility that once the cells expressingthese receptors are generated, their survival and growth (“selec-tion”) may be dependent upon Ag; the identity of these Ags has notbeen established. Specific fetal receptors could also be maintainedthroughout life as a consequence of their continuous generationand/or longevity. The continuous de novo generation of fetal re-ceptors seems less likely, since the mechanisms that diversifyAgRs also become more active and are extensively used to gen-erate diverse repertoires as ontogeny proceeds. Their longevitymay be due to continuous or periodic exposure to Ag. For exam-ple, the expansion of DV2R-bearinggd T cells that is seen in theperipheral blood of neonates (52, 53) suggests that the initial ex-posure to environmental Ags is one such timepoint during whichfetal or neonatally derivedgd T cells can be expanded. The pos-sibility that the retention ofgd T cell subsets in specific tissuemicroenvironments may also contribute to their longevity is sug-gested by the finding that the survival of Ag-stimulated T cells isincreased as a result of their ability to bind and interact with tissuefibroblasts (54).

Our comparative analyses of TCRDVR expression in fetal he-matopoietic tissues clearly distinguish the intestinal repertoirefrom that of the liver and thymus. In addition, our results also showthat the oligoclonality of TCRDVRs, which is a hallmark featureof the intestinalgd T cell repertoire in humans (9–15), is notestablished until very late (after 33 wk) in gestation or, as sug-gested by others (15), after birth. Although experiments conductedin conventionalized germfree mice suggest that exposing mucosalT cells to commensal gut bacteria can influence the repertoire ofAg and TCR specificities present (55), and that DV1-expressinghuman cord blood T cells can proliferate in vitro in the presence ofcertain bacterial Ags (56), there is no compelling evidence that thismechanism “shapes” the intestinal TCRD repertoire in humans.

A comparison of TCRDV1R and TCRDV2R sequences in 15 wkfetal thymus and gut samples from the same donor indicated that therewere no overlapping sequences shared between these sites; the phe-notype of intestinal and thymicgd T cells is distinct and is consistent

with functionally distinct receptor-bearing cells residing in differentcompartments. In addition, a comparison of DV1R and DV2R se-quences present in the gut of the same donor at mid-gestation (15 wk)and late gestation (33 wk), showed that each receptor was processeddifferently, resulting in receptors that have extensive (DV1) or mini-mal (DV2) receptor sequence diversity. These differences in the pro-cessing of gut DV1Rs and DV2Rs may be attributable to differencesin the availability or accessibility of each TCRDR to the machineryresponsible for diversifying AgRs, such as exonucleases and terminaldeoxynucleotidyltransferase. This may be a general feature in normalfetal development, since the differential processing of rearrangedTCRD receptors found in the fetal gut has previously been identifiedin the human fetal thymus (27, 57). The mechanisms that generateAgR diversity appear, therefore, to operate differently for distinctTCRDV-DCRs.

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

References1. McVay, L. D., and S. R. Carding. 1996. Extrathymic origin of humangd T cells

during fetal development.J. Immunol. 157:2873.2. Wuchferpennig, K. W., Y. J. Liao, M. Prendergast, J. Prendergast, D. Hafler, and

J. Strominger. 1993. Human fetal liverg/d T cells predominantly use unusualrearrangements of the T cell receptord andg loci expressed on both CD41CD82

and CD42CD82 g/d T cells.J. Exp. Med. 177:425.3. Sanchez, M. J., J. C. Gutierrez-Ramos, E. Fernandez, E. Leonardo, J. Lozano, C.

Martınez-A, and M. L. Toribio. 1993. Putative prethymic T cell precursors withinthe early human embryonic liver: a molecular and functional analysis.J. Exp.Med. 177:19.

4. WHO-IUIS Nomenclature Sub-Committee on TCR Designation. 1995. Nomen-clature for T-cell receptor (TCR) gene segments of the immune system.Immu-nogenetics 42:451.

5. Spencer, J., P. G. Isaacson, T. C. Diss, and T. T. MacDonald. 1989. Expressionof disulfide-linked and non-disulfide-linked forms of the T-cell receptorg/d het-erodimer in human intestinal intraepithelial lymphocytes.Eur. J. Immunol. 19:1335.

6. Halstensen, T. S., H. Scott, and P. Brandtzaeg. 1989. Intraepithelial T cells of theTCRg/d1 CD82 and Vd1/Jd11 phenotypes are increased in coeliac disease.J. Immunol. 30:665.

7. Ullrich, R., H. L. Schieferdecker, K. Zeigler, E. O. Reicken, and M. Zeitz. 1990.g/d T cells in the human intestine express surface markers of activation and arepreferentially located in the epithelium.Cell. Immunol. 128:619.

8. Deusch, K., F. Luling, K. Reich, M. Classen, H. Wagner, and K. Pfeffer. 1991.A major fraction of human intraepithelial lymphocytes simultaneously expressesthe gd T cell receptor, the CD8 accessory molecule and preferentially uses theVd1 gene segment.Eur. J. Immunol. 21:1053.

9. Chowers, Y., W. Holtmeier, J. Harwood, E. Morzycka-Wroblewska, andM. F. Kagnoff. 1994. The Vd1 receptor repertoire in human small intestine andcolon.J. Exp. Med. 180:183.

10. Holtmeier, W., Y. Chowers, A. Lumeng, E. Morzycka-Wroblewska, andM. F. Kagnoff. 1995. Thed T cell repertoire in human colon and peripheral bloodis oligoclonal irrespective of V region usage.J. Clin. Invest. 96:1108.

11. Balk, S. P., E. C. Ebert, R. L. Blumenthal, F. V. McDermott,K. W. Wuchferpennig, S. B. Landau, and R. S. Blumberg. 1991. Oligoclonalexpansion and CD1 recognition by human intestinal intraepithelial lymphocytes.Science 253:1411.

12. Van Kerckhove, C., G. J. Russell, K. Deusch, K. Reich, A. K. Bhan,H. DerSimonian, and M. B. Brenner. 1992. Oligoclonality of human intestinalintraepithelial T cells.J. Exp. Med. 175:57.

13. Blumberg, R. S., C. E. Yockey, G. G. Gross, E. C. Ebert, and S. P. Balk. 1993.Human intestinal intraepithelial T lymphocytes are derived from a limited num-ber of T cell clones that utilize multiple Vb TCR genes.J. Immunol. 150:5144.

14. Gross, G. G., V. L. Schwarz, C. Stevens, E. C. Ebert, R. S. Blumberg, andS. P. Balk. 1994. Distribution of dominant T cell receptorb chains in humanintestinal mucosa.J. Exp. Med. 180:1337.

15. Holtmeier, W., T. Witthoft, A. Hennemann, H. S. Winter, and M. F. Kagnoff.1997. The TCR-d repertoire in human intestine undergoes characteristic changesduring fetal to adult development.J. Immunol. 158:5632.

16. Lin T., G. Matsuzaki, H. Yoshida, N. Kobayashi, H. Kenai, K. Omoto, andK. Nomoto. 1994. CD32CD81 intestinal intraepithelial lymphocytes (IEL) andthe extrathymic development of IEL.Eur. J. Immunol. 24:1080.

17. Kanamori, Y., K. Ishimaru, M. Nanno, K. Maki, K. Ikuta, H. Nariuchi, andH. Ishikawa. 1996. Identification of novel lymphoid tissues in murine intestinal

5859The Journal of Immunology

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

mucosa where clusters of c-kit1 IL-7R1 Thy-11 lympho-hematopoietic progen-itors develop.J. Exp. Med. 184:1449.

18. Mosely, R. L., and J. R. Klein. 1992. Peripheral engraftment of fetal intestine intoathymic mice sponsors T cell development: direct evidence for thymopoieticfunction of murine small intestine.J. Exp. Med. 176:1365.

19. Poussier, P., H. S. Teh, and M. Julius. 1993. Thymus-independent positive andnegative selection of T cells expressing a major histocompatibility complex classI-restricted transgenic T cell receptora/b in the intestinal epithelium.J. Exp.Med. 176:1947.

20. Rocha, B., H. von Boehmer, D. Guy-Grand. 1992. Selection of intraepitheliallymphocytes with CD8a/a co-receptors by self-antigen in the murine gut.Proc.Natl. Acad. Sci. USA 89:5336.

21. Poussier, P., P. Edouard, C. Lee, and M. Julius. 1992. Thymus-independent de-velopment and negative selection of T cells expressing T cell receptora/b in theintestinal epithelium: evidence for distinct circulation patterns of gut- and thy-mus-derived T lymphocytes.J. Exp. Med. 176:187.

22. Golunski, E., and R. Palacios. 1994. Fetal liver and bone marrow JORO 751

lymphocyte progenitors are precursors of CD41CD82TCR/CD32 early thymo-cytes.J. Exp. Med. 179:721.

23. Vidal, K., I. Grosjean, J. P. Revillard, C. Gespach, and D. Kaiserlian. 1993.Immortalization of mouse intestinal epithelial cells by the SV40-large T gene.J. Immunol. Methods 166:63.

24. Klein, J. R. 1995. T-lymphopoietic capacity of the mouse intestinal epithelium.Semin. Immunol. 7:291.

25. Streeter, G. L. 1948. Developmental horizons in human embryos: description ofage groups XV, XVI, XVII, and XVIII.Contrib. Embryol. Carnegie Inst. 32:133.

26. O’Rahilly, R. 1972. Guide to the staging of human embryos.Anat. Anz. 130:556.27. McVay, L. D., S. R. Carding, K. Bottomly, and A. Hayday. 1991. Regulated

expression and structure of T cell receptorg/d transcripts in human thymic on-togeny.EMBO J. 10:83.

28. Carding, S. R., S. Kyes, E. J. Jenkinson, R. Kingston, K. Bottomly, J. J. T. Owen,and A. Hayday. 1990. Developmentally regulated fetal thymic and extrathymicT-cell receptorgd gene expression.Genes Dev. 4:1304.

29. Carding, S. R., J. G. McNamara, M. Pan, and K. Bottomly. 1990. Characteriza-tion of gd T cell clones isolated from human fetal liver and thymus.Eur. J. Im-munol. 20:1327.

30. Moore, K. L. 1982.The Developing Human—Clinically Oriented Embryology.W. B. Saunders Company, Philadelphia, Ch. 12, pp. 227–254.

31. Orlic, D., and R. Lev. 1977. An electron microscopic study of intraepitheliallymphocytes in human fetal intestine.Lab. Invest. 37:554.

32. Spencer, J., P. G. Isaacson, J. A. Walker-Smith, and T. T. MacDonald. 1989.Heterogeneity in intraepithelial lymphocyte subpopulations in fetal and post-natalhuman small intestine.J. Pediatr. Gastroenterol. Nutr. 9:173.

33. MacDonald, T. T., and J. Spencer. 1994. Ontogeny of the gut-associated lym-phoid system in man.Acta Paediatr. Suppl.83:(395):3.

34. Kabelitz, D., and P. Conradt. 1988. Identification of CD22/CD31 T cells inhuman fetal tissue.J. Exp. Med. 168:1941.

35. Takihara, Y., D. Tkachuk, E. Michalopoulos, E. Champagne, J. Reimann,M. Minden, and T. W. Mak. 1988. Sequence and organization of the diversity,joining, and constant region genes of the human T-celld-chain locus.Proc. Natl.Acad. Sci. USA 85:6097.

36. Takihara, Y., J. Reimann, E. Michalopoulos, E. Ciccone, L. Moretta, and T. Mak.1989. Diversity and structure of human T cell receptord chain genes in peripheralblood g/d-bearing T lymphocytes.J. Exp. Med. 169:393.

37. Klein, M. H., P. Concannon, M. Everett, L. D. H. Kim, T. Hunkapillar, andL. Hood. 1987. Diversity and structure of human T-cell receptora-chain variableregion genes.Proc. Natl. Acad. Sci. USA 84:6884.

38. Wright, J. A., L. Hood, and P. Concannon. 1991. Human T-cell receptor Va genepolymorphism.Hum. Immunol. 32:277.

39. Schatz, D. G., M. A. Oettinger, and D. Baltimore. 1989. The V(D)J recombina-tion activating gene, RAG-1.Cell 59:1035.

40. Oettinger, M. A., D. G. Schatz, C. Gorka, and D. Baltimore. 1990. RAG-1 andRAG-2, adjacent genes that synergistically activate V(D)J recombination.Sci-ence 248:1517.

41. Triebel, F., F. Faure, F. Mami-Chouaib, S. Jitsukawa, A. Griscelli, C. Genevee,S. Roman-Roman, and T. Hercend. 1988. A novel human Vd gene expressedpredominantly in the TigA1 fraction ofgd1 peripheral lymphocytes.Eur. J. Im-munol. 18:2021.

42. Casorati, G., G. De Libero, A. Lanzavacchia, and N. Migone. 1989. Molecularanalysis of humang/d1 clones from thymus and peripheral blood.J. Exp. Med.170:1521.

43. Bottino, C., G. Tambussi, S. Ferrini, E. Ciccone, P. Varese, M. C. Mingari,L. Moretta, and A. Moretta. 1988. Two subsets of human T lymphocytes ex-pressingg/d antigen receptor are identifiable by monoclonal antibodies directedto distinct molecular forms of the receptor.J. Exp. Med. 168:491.

44. Borst, J., J. J. M. van Dongen, R. L. H. Bolhuis, P. J. Peters, D. A. Hafler,E. de Vries, and R. J. van de Griend. 1988. Distinct molecular forms of humanT cell receptorg/d detected on viable T cells by a monoclonal antibody.J. Exp.Med. 167:1625.

45. Bodger, M. P., G. Janossy, F. J. Bollum, G. D. Burford, and V. Hoffbrand. 1983.The ontogeny of terminal deoxytidyl transferase positive cells in the human fetus.Blood 6:1125.

46. Mincheva-Nilsson, L., M. Kling, S. Hammarstrom, O. Nagaeva, K.-G. Sundqvist,M.-L. Hammarstrom, and V. Baranov. 1997.gd T cells of human early pregnancydecidua: evidence for local proliferation, phenotypic heterogeneity, and extrathy-mic differentiation.J. Immunol. 159:3266.

47. Cepek, K. L., S. K. Shaw, C. M. Parker, G. J. Russell, J. S. Morrow, D. L. Rimm,and M. B. Brenner. 1994. Adhesion between epithelial cells and T lymphocytesmediated by E-cadherin and theaE/b7 integrin.Nature 372:190.

48. White, B., J. H. Korn, and T. H. Piela-Smith. 1994. Preferential adherence ofhumangd, CD81, and memory T cells to fibroblasts.J. Immunol. 152:4912.

49. McVay, L. D., B. Li, R. Biancaniello, M. A. Creighton, D. Bachwich,G. Lichtenstein, J. L. Rombeau, and S. R. Carding. 1997. Changes in humanmucosalgd T cell repertoire and function associated with the disease process ininflammatory bowel disease.Mol. Med. 3:183.

50. Davodeau, F., M. A. Peyrat, M. M. Hallet, I. Houde, H. Vie, and M. Bonneville.1993. Peripheral selection of antigen receptor junctional features in a major hu-mangd subset.Eur. J. Immunol. 23:804.

51. Migone, N., S. Padovan, C. Zappador, C. Giachino, M. Bottaro, G. Matullo,C. Carbonara, G. De Libero, and G. Casorati. 1995. Restriction of the T-cellreceptor V delta gene repertoire is due to preferential gene rearrangement and isindependent of antigen selection.Immunogenetics 42:323.

52. Parker, C. M., V. Groh, H. Band, S. Porcelli, C. Morita, M. Fabbi, D. Class,J. L. Strominger, and M. Brenner. 1990. Evidence for extrathymic changes in theT cell receptorgd repertoire.J. Exp. Med. 171:1597.

53. Borst, J., A. Wicherink, J. J. M. van Dongen, J. E. de Vries, W. H. Comans-Bitter,F. Wassenaar, and P. van den Elsen. 1989. Non-random expression of T-cellreceptorg andd variable gene segments in functional T lymphocyte clones fromhuman peripheral blood.Eur. J. Immunol. 19:1559.

54. Scott, S., F. Pandolfi, and J. T. Kurnick. 1990. Fibroblasts mediate T cell survival:a proposed mechanism for retention of primed T cells.J. Exp. Med. 172:1873.

55. Umesaki, Y., H. Setoyama, S. Matsuomo, Y. Okada. 1993. Expansion of alphabeta T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbialcolonization in germ-free mice and its independence from the thymus.Immunol-ogy 79:32.

56. Kersten, C. M., R. T. McCluskey, L. A. Boyle, and J. T. Kurnick. 1996.Esch-erichia coli andPseudomonas aeruginosainduce expansion of Vd2 cells in adultperipheral blood, but of Vd1 cells in cord blood.J. Immunol. 157:1613.

57. Krangel, M. S., H. Yssel, C. Brockelhurst, and H. Spits. 1990. A distinct wave ofhumangd T lymphocytes in the early fetal thymus: evidence for controlled generearrangement and cytokine production.J. Exp. Med. 172:847.

5860 HUMAN gd T CELL DEVELOPMENT

by guest on May 18, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from