Regulation of the mature human T cell receptor gamma repertoire by biased V J gene rearrangement

Regulation of the mature human T cell receptor

gamma repertoire by biased V-J gene

rearrangement.

H Kohsaka, … , W E Ollier, D A Carson

J Clin Invest.

1993;

91(1)

:171-178.

https://doi.org/10.1172/JCI116167

.

To delineate how gene rearrangement influences the expressed human gamma delta T cell

repertoire, we generated T cell receptor gamma (TCR gamma) V domain-specific cDNA

libraries from the peripheral lymphocytes of eight donors and sequenced a total of 232 TCR

gamma gene transcripts. The libraries consisted of both in-frame and out-of-frame

rearranged TCR gamma genes. The in-frame TCR gamma gene transcripts were used to

determine the diversity of functional T cells, whereas the out-of-frame transcripts, primarily

derived from alpha beta T cells, were used to assess the frequencies of TCR V gamma-J

gamma rearrangements in progenitor T lymphocytes. The results showed that both sets of

transcripts exhibited strikingly restricted V gamma-J gamma combinations. Only 11 of 40

potential V gamma-J gamma rearrangements were common ( > or = 3% of total). The pattern

of gene usage in the functional and nonfunctional transcripts was similar and did not differ

markedly among donors. The only exception was the predominance of V gamma 9-JP in

potentially functional transcripts from seven of eight individuals. These results show that V

gamma-J gamma rearrangement is nonrandom and suggest that the diversity of TCR

gamma genes in the functional gamma delta T cell repertoire partly depends upon

preferentially rearranged V gamma-J gamma gene combinations. However, the expansion

of V gamma 9/V gamma 2 T cells in adult […]

Research Article

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Regulation of the Mature Human

T

Cell

Receptor

y

Repertoire by Biased V-J Gene Rearrangement

Hitoshi Kohsaka, PojenP.Chen, Atsuo Taniguchi,William E. R.Ollier,*and Dennis A. Carson

TheDepartmentofMedicine and theSamandRoseSteinInstituteforResearchonAging, UniversityofCalifornia,SanDiego,LaJolla,

California 92093;and *Arthritis and Rheumatism CouncilEpidemiologyResearch Unit, University ofManchester, Manchester, UnitedKingdom

Abstract

Todelineate how gene rearrangement influences the expressed human

'y

T cell repertoire, we generated T cell receptor

"y

(TCRy)Vdomain-specificcDNAlibraries from the peripheral

lymphocytes of eight donors and sequenced a total of 232

TCR-t

gene transcripts. The libraries consisted of both in-frame and out-of-in-frame rearranged

TCRy

genes.The in-frame

TCRy

genetranscriptswere used todeterminethediversity of functional T cells, whereas the out-of-frame transcripts, pri-marily derivedfrom a,B T cells, were used to assess the frequen-cies of TCR V-y-Jy rearrangements inprogenitor T lympho-cytes. The results showed that both sets oftranscripts

exhib-ited strikinglyrestricted V-y-J-y combinations. Only 11 of 40

potential

V-y-J'y

rearrangements were common (23% of

to-tal).The patternofgene usage in thefunctionaland nonfunc-tional transcripts was similar and did not differ markedly among donors. Theonly exception was the predominance of Vy9-JP in potentially functional transcripts from seven of

eight

individuals. These results show that

V-y-Jy

rearrange-ment is nonrandom and suggest that the diversity of

TCRy

genes in thefunctional

'y

Tcellrepertoirepartlydependsupon

preferentiallyrearranged

Vy-J'y

genecombinations.However, theexpansion of

Vy9

/V62Tcellsinadultperipheral bloodcan only beexplainedbyantigenic selectionofrelativelyrare

V'y9-JP recombinants. (J. Clin. Invest. 1993. 91:171-178.) Key words: generecombination * adult peripheral T cell * anchored

polymerasechainreaction*biasedV-J gene usage * nonfunc-tionaltranscript

Introduction

Tlymphocytesexpress twomutually exclusivetypesof antigen

receptor,either af3or

-ye,

in association with theCD3

molecu-lar complex. The smaller y subsetofTcellshas beenfound in all vertebratespecies examined. Most

'y

cells do not express

CD4 orCD8accessory moleculeson their

surface,

andtheir recognition of antigen isnotrestrictedbyclassicalMHC mole-cules(1). Murine

'y

Tcells have adistinctive ontogenyand tissue distribution. Thepresenceoftwoclassesof murine

'yS

T

Addresscorrespondence to Hitoshi Kohsaka, M.D., Department of Medicine 0663, University ofCalifornia, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663.

Receivedfor publication 24 March 1992 and in revised form 13 August1992.

cells, i.e.,intraepidermal lymphocyteswith restricted V'y/Vb

receptors and secondary lymphoid organ lymphocytes with

variable

Vy

/VA receptors, suggest that these two types of cells

mightfulfill special roles in the immunesystem(2).Humany

Tcells donothave thesametissuetropism. The physiological functions of murineand human

_yb

Tcells are still unclear.

Asmallsetof V, D, and Jgenesegmentsencode human

ye

receptors. This limiteddiversity makes it possibleto assessthe factorsthatcontroldevelopmentandexpressionof the mature 'ye T cell

repertoire.

By analogy

with a13 T cell

receptor

(TCR),'these couldincludepreferentialgenerecombination, positive and

negative

selection by self-antigensinthethymus,

andperipheral expansionof T cell clonesby antigens.Todate,

the adult 'ye T cell repertoire has been studied mainly with monoclonalantibodies.Twodominant subsetsof human

yS

T cells have been recognizedin peripheral blood (3). Southern blotanalyses ofgenomicDNAfrom clonedlymphocytesfrom each subsetindicatethat oneclassuses

V'y9-JP-Cy

1

/VV62-Je

1-Ce 1 toform the TCR heterodimer whereas the otherusesV'y2

or

Vy4-J2-Cy2/

VAI-Je I-CA1 (4).

Evidence for

regulation

of

TCRy

gene rearrangement in early ontogeny has comefrom analyses of

ye

transcriptsin the

fetal and neonatal

lymphoid

tissues (5, 6). However,whether

selective rearrangement of TCR-y genes influences the adult

expressedhuman

-ye

T cellrepertoireis not known. One wayto

determine the effect of intrinsic gene rearrangement on the

expressed

repertoire

istocomparethefrequenciesof all

poten-tial Vy-Jy rearrangements in functional and nonfunctional

TCR Vy genes. This ispossible because the

TCRy

cDNAin

peripheral

lymphocytes includes both functionally and

non-functionally rearranged

transcripts

(7).

We,

therefore,

used the anchoredpolymerase chain

reac-tion

(PCR)

method

(8)

to construct

TCRy

cDNA libraries

fromthe peripheral blood

lymphocytes

of eight

individuals,

compared gene

frequencies

amongthe in-frame and out-of-frame TCR^ytranscripts,andsequencedatotalof232

V-y-J^y

genes. Except for

V-y9-JP-C'y

1, both functional and nonfunc-tionalgenesexhibitedasimilarly restrictedsetof

Vy-Jy

combi-nations. We conclude that regulation of TCR V^ygene rear-rangementisa significant forceinmoldingthe mature func-tional

-ye

Tcell

repertoire.

However,

antigenic selection,

asin

the caseof

V-y9-JP-bearing

cells,can

potentially

expandeven very rare recombinants.

Methods

Bloodsamples. PBMCswereobtained from eight donors. Their ages rangedfrom 39 to65yr.Mononuclearcells wereisolatedbydensity

1.Abbreviations usedinthis paper: PCR, polymerase chain reaction; TCR,T cellreceptor.

J.Clin. Invest.

©The American Society for Clinical Investigation, Inc. 0021-9738/93/01/0171/08 $2.00

centrifugationand werestored in medium containing 10% DMSOat

-80°C until used.

RNA isolation andfirst-strandcDNA synthesis. Total RNA was isolated from 5 x 105 PBMCs using RNAzolB (Cinna/Biotecx, Friendswoods, TX), which is based onthe acid guanidium

thiocya-nate-phenol-chloroform method(9). First-strand cDNAwas synthe-sizedfrom totalRNAusinganoligo-dT primer and Superscriptreverse

transcriptase(Life Technologies, Inc., Gaithersburg, MD). The result-ing cDNA/RNA hybridswerefirst size selected (0.5-3 kb)onagarose

gels and extracted with phenol. The remainingRNA washydrolyzed by incubationat60°C in 0.3MNaOH.

dGtailing

offirst-strand

cDNA and anchored PCR. Toamplifyan entireTCRy V domain cDNA segment regardless ofsequence, the

first-strand cDNAwaspoly dG tailed with dGTP and terminal deoxy-transferase. Subsequently free dGTPwasremovedbytwosequential ammonium acetate precipitation steps. The G-tailed first-strand

cDNA wassubjectedtoprimary anchored PCR amplification in 100,ul

of 10mM Tris-HCl (pH 8.3), 50mM KCI, 2 mM MgCl2, 0.001% gelatin, 200 ,uM dNTPs, 1 U Perfect Match Polymerase Enhancer (Stratagene, Inc., La Jolla, CA), and 2.5UAmpliTaqDNA

polymer-ase (Perkin-Elmer Cetus Instruments, Norwalk, CT). The primers consisted ofaCy-specificantisenseoligonucleotide(Cyb:

5'-CACG-GTCGACTCAAGAAGACAAAGGTATGT) and anchor primers

(9:1 mixture ofANprimer: 5'-ATTACGGCGGCCGCGGATCC and ANC primer: 5'-ATTACGGCGGCCGCGGATCCCCCCCCCCCC-CC), at a concentration of 1 MM. The ratio of AN 1 and AN1C

was based on the report by Loh et al. (8). The anchor primers

containedsequences thatarerecognized byrestriction enzymeNotI

(GCGGCCGC) and BamHI (GGATCC), respectively, whereas the

C'ybprimerhadasequencerecognized by SalI(GTCGAC)atits 5' end. These restriction sites facilitated the subsequent subcloning of the PCR products. The amplification included 20 cycles of 95°C for 30s,

42°C for 30s,and72°C for1 minfollowed by final extension of7min.

Theproductsweresize selected (500-750 bp)onagarosegels andone

third were removed for nested PCR. The reaction mixturewasthe

same asthat oftheprimary anchored PCRexceptfor theprimers, 14M

eachofANprimer and antisenseCyaprimer ( 5'-ACGCGTCGACGG-AAGAAAAATAGTGGGCTT, which containedaSalIrecognition se-quenceatits5'end, andprimesupstreamof the Cybprimer sequence)

and theconcentration ofMgCl2(1.5 mM). 20 cycles of nested PCR

werecarriedout atanannealingtemperatureof 55°C, and the products

werepurifiedby phenol extraction.

ConstructionofTCRy-specificcDNAlibraries. A short fragment of the multicloning site of pBluescriptIISK+ (Stratagene,Inc.) was

re-leased by enzymatic digestion withSacd and Sal Irestrictionenzymes andinserted into the polylinker ofthe M 13mp19 vector, to produce a M13vectorthatincludes SalIandNotIsites. The PCRproducts were

digestedwith SalIandNotItomakecohesive ends,andfragments of theappropriatesizewereseparatedonagarosegels andextractedwith phenol.Thepurified cDNAwasligated with the SalI/NotI-digested

M13vectorand theligatedvector waselectrotransformed into SURE Escherichia coli (Stratagene, Inc.).

Screeningandfrequencyanalysis of

TCR-y-specific

cDNAlibraries.

M13 plates, with plaques containing amplified TCR-y V domain cDNA, were lifted five times with Hybond-N+ nylon membranes

(Amersham Corp., Arlington Heights, IL).Both aninternalCyprobe

(Cyc: 5'-TAAACAACTTGATGCAGATG, recognizingaregion

up-stream of theCya primer-primingsequence)andVyfamily-specific probes (V-yI: 5'-GTCAGAAATCTTCCAACTTGGAAG, VyII:

5'-GACGGCACTGTCAGAAAGGAATCT, VyIII: 5'-TCAGGCTTTG-GAGCACCTGATCT, and VyIV: 5'-CAAAGGCTTAGAATATT-TAT)werelabeledwithdigoxigeninin 20Mlof 300 pmol oligonucleo-tide,200 mMpotassium cacodylate,25 mMTris-HCl (pH 6.6), 1.5

mMCoCI2,0.25mg/ml bovineserumalbumin, 125,M

digoxigenin-1l-dUTP(Boehringer Mannheim Biochemicals, Indianapolis, IN), 5

,MMdATP, and 50Uterminaldeoxytransferase.Fivemembranes were

hybridized withthedifferent probesat42°C. Afterwashing the

mem-branes, positive plaques were visualized using the Genius system (Boehringer Mannheim Biochemicals). Briefly, the membraneswere

blocked with blocking reagent and incubated with antidigoxigenin an-tibody conjugated with alkaline-phosphatase. Then they were soaked in an adamantyl 1,2-dioxetane phosphate (AMPPD; Tropix, Inc., Bed-ford, MA) solution and incubated at 37°C to activate the phosphatase. Positive signals were recorded on x-ray film and enumerated.

Conventional PCR. The ability of the VyIV PCR primer to amplify rearranged Vy 11 genes (from the VyIV family) was confirmed by con-ventional PCR. Template DNA was isolated from PBMCs. Amplifica-tion was carried out using 100 Ml of 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.001% gelatin, 200MMdNTPs, 2.5 U AmpliTaq DNA polymerase, and 1 MuM of theVyIVfamily-specific sense oligonu-cleotide and either theCy-specificantisense oligonucleotides(Cybor Cyc: 5'-ATCTGCATAAGTTGTTTA primer without restriction en-zyme site) or a J1,J2-specific antisense oligonucleotide (J 12:5'-GTGTTGTTCCACTGCCAAAG). The PCR products were visual-ized after separation by agarose gel electrophoresis.

Enrichment ofa(x T cell or-ybT cellpopulations. PBMCs from one donor were incubated at 2x 107 cells for 30 min at 4°C with FITC-conjugated mouse anti-TCR6 chain antibody (TCR6 1; T Cell Sciences, Inc., Cambridge, MA) at 0.8,g/mlin 0.8 ml of RPMI 1640 medium supplemented with 10% bovine serum albumin. Cells were washed twice and incubated for 30 min at 4°C with 0.3 mg of magnetic beads conjugated to sheep anti-mouse IgG (DYNAL, Inc., Great Neck, NY). After washing and magnetic removal of the beads, cell-coated beads were used directly for RNA extraction of cells enriched inybT lymphocytes. The remaining cells were further incubated with 3 mg of thebeads to maximize the removal ofybTcells and bound cells were removed by magnetic separation. The final cell suspension was used for RNA extraction of cells enriched ina/3T lymphocytes. Cytofluorome-tric analysis showed that the enrichedafiT cellpopulation contained 0.3% yb cells compared with 6% in unfractionated PBMCs.

DNAsequencing. Replica form double-strand DNA and/or single-strand phage DNA were extracted from bacterial cultures contain-ing recombinant M 13 and used as sequencing templates. Sequenc-ing was carried out with the dideoxynucleotide chain-termination method(10).

Results

TCR'y

Vdomaingene repertoire ofpotentially functional and

nonfunctional transcripts. Of the 14 reported human

Vy

genes, 8 arepotentially functional, whereas6 are pseudogenes. The

eight potentially functionalgenes aredivided into four

fami-lies.The VyI family includes five functionalgenes(Vy2, V'y3,

Vy4, Vy5, Vy8), whereas the three other Vy families have

onlyonefunctional member (Vy9, Vy10,and V-y 1 1,

respec-tively). Four oligonucleotides(V-yl, VyII, VyIlll, andV'yIV)

were designedto specify members ofeachcorrespondingVy

family. Poly G-tailed cDNAsweresynthesized from mRNAs

ofPBMCs obtained from eight donors. Expressed Vy genes wereamplified from thecDNAsusing primaryanchoredPCR and nested PCR. After TCRy-specific cDNA librarieswere

con-structed in a modified M13 vector, they were screened with bothaCyprobe andVyfamily-specificprobes. Gene frequen-cieswerecalculated bydividingthe numberofVyfamily posi-tiveclones by the numberoftotalTCRyclones.

Inthe firstsetof experiments, the reproducibility and fidel-ity of the amplification methodwasvalidated. cDNA obtained fromonedonorwassubjectedtothree different PCR amplifi-cations. The calculated frequency datawereconservedamong

different experiments and varied by <5%.

In eight separate TCRy gene-specific libraries from eight

donors,nocloneswerepositive with the VyIV family-specific probe. Approximately 10 clones from the set ofeach

Vy

family positive plaques were randomly selected and a total of 232

V-y-Jy-C,y

geneswere sequenced. Allgenescould be classified according to the usage ofJychains. Since the

VyI-family

in-cludes five functional genes,

Vyl

subgroup positive clones were further classified by the usage of

Vy

genes. Although the

Cy-specific

primers used for amplification and probing were complimentary to both the

Cy

1 gene and the

Cy2

gene, the

Cy

chain usage was deduced from the known

Jy

segment configu-rations (11).

The potential functionalityof each clone wasdetermined byexamining the sequence at the V-J junction for rearrange-mentsmaintaining the correct reading frame. Representative

TableI.Representative FrequencyAnalysesofIndividualDonors

DonorA Vyl Vyll (V-y9) V4III(Vy10)

Frequency in totaltranscripts 26.3 39.9 33.8 Frequency inpotentially

functionaltranscripts 27.0 52.2 20.8

Frequency in nonfunctional

transcripts 25.6 28.3 46.1

JC segment usage VYI VYII VYIII

(n= 10) (n= 11) (n= 10)

Translationalframe In Out In Out In Out

JPI-CY1 2* 2 2

JP-C'yl 6

J

1-C71

2§ 1

JP2-Cy2

J2-C72

3* 311 1 4 1 4

*Vy8(2). tVy3,Vy5, Vy8.

tVy4,

Vy5. 11_Vy2(2),Vy3.

DonorB VyI V-rI (V-y9) VyIII (Vy10)

Frequency in totaltranscripts 29.0 3.0 68.1 Frequency inpotentially

functionaltranscripts 51.0 1.2 47.9

Frequency in nonfunctional

transcripts 0 5.4 94.6

JC segment usage VYI V-II VyIiI

(n=8) (n=9) (n=10)

Translational frame In Out In Out In Out

JPI-Cyl

2* 2 5

JP-C' Il

J1-C'y1 2 1

JP2-Cy2

JP-Cy2 6* 1 7

*Vy2(2). *_{Vy2,Vy3, Vy8(4).}

resultsfromthree donors (A, B, andC)areshown in Table I. Although the

TCRy

repertoires variedamongdonors,a

simi-larlyskewed pattern of

Vy-Jy

combinationswasobservedinall

cases.

The V-Jjunctional sequences ofamplified

TCRy

cDNA from the same donor are shown in Table II.Anextensive

num-ber of differentsequencesatthe

V-y-Jy

junction (N region)was

observed. Onlyasingle pair ofidentical sequenceswasfound among30 separate clones.These data show that the PCR

am-plificationwasperformed without preferential amplification of particularsequences.

Average

frequencies of

transcripts

classified by

Vy and Jy chain usage. Onthebasis ofthefrequencyandjunctional se-quence dataofVy familygenes, thefrequencyof eachVy-Jy

DonorC VyI V-yII(Vy9) VyITI(V 10)

Frequency in totaltranscripts 48.8 41.5 9.6 Frequency inpotentially

functionaltranscripts 41.0 52.0 7.0

Frequency innonfunctional

transcripts 63.4 20.0 16.6

JC segment usage Vyl V'yII VYIII

(n= 10) (n=9) (n=10)

Translational frame In Out In Out In Out

JPl-CYl 1* 1 2

JP-Cy1 6

Jl-CyI

JP2-Cy2

J2-C,y2

6*

3§ 2 1 5 2

*_{Vy4. $Vy2(3),V-y3, V-y4,Vy8;} §_V-y8(3).

The frequencies of expression of theVyl,VyllandVyIII gene fami-lies in the librarieswerecalculatedby enumeratingpositiveplaques of eachVyfamily,comparedtothe totalCypositiveplaques. Then,

10 clones from each Vyfamilywerepickeduprandomly,

+ + + + + + + + + + + + + +

C-1 - I.- - ,1 - C-) - -1 I.- , _I.-, C- I--, C-C C- CI- C-) C-) `-)

C-CDCDCD O CD U CD _H F, H

EqH000000vE- E- ¢ ¢ _H E_EHHH E-vE

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EOH 0E¢

¢¢¢E-4 E- ¢E

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HE-H

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EH E- H E OO OU

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000000 E)0E0

¢ ¢ ¢ ¢OHHH

<<<<<<<000O C OOHDHDCOO)C

OOO)CDO OOOHHHC

E00q 0v E0 C0OOC

E- E E-4 ¢ <4EqEvE vE _E- ¢

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CD4 U C C C E-4

¢ H HH¢

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¢H ¢H¢ HH¢H¢

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TableIII. AverageFrequencies ofTranscriptsfor Each V-y-J'y Combination from EightDonors

Vygene

J-y-Vycombination V-y2 V-y3 V-y4 V-y5 V-y8 VY9 V-y10

Potentially functional transcripts

JPI-CY1 1.5 0 1.0 0 1.1 0 5.3

JP-CyI 0 0 0 0 0 25.3 0

Jl-C'yl 0 7.0 0 0 0 0.4 4.1

JP2-Cy2 0 0 0.6 0 0 0 0

J2-C-y2 6.6 3. 1 8.2 1.4 9.2 8.9 16.6

Nonfunctionaltranscripts

JPI-CYl 0 0 0 0 0 0 17.8

JP-CyI 0 0 0 0 0 0.9 0

Jl-CYl 0 0 0.9 0.9 0 1.0 13.9

JP2-C,y2 0.6 1.1 0 0 0 0 0

J2-Cy2 9.7 3.5 4.1 4.6 5.8 13.1 22.1

Averagefrequencies of the potentially functional transcripts (upper) and nonfunctional transcripts (lower) for eachV-y-Jy-Cycombinationare

shown.The data arefromeight individuals, includingthe donorsshown in Table I. Thefrequencyof eachVygenefamilywasdeterminedby

plaquehybridizationin alleight libraries; and then individual gene usage and gene functionalitywasassessedby sequence analysis of 10 clones from each Vy family, in all eight libraries. Finally, the average frequencies for theV-y-Jycombinationswerecalculated.

combinationwasdeterminedinthepotentiallyfunctional and

nonfunctional repertoire ofeach donor.Since the basicpattern

of

_Vy-Jy

combinationswassimilaramongdonors,the average

frequencies of transcripts with eachVy-J-y combination were calculated(Table III).Inboth the in-frametranscriptsand the

out-of-frametranscripts,abiased increaseofparticular

Vy

and

J-y pairs

wasobserved. The J2genesegmentwasused in 54% of the in-frametranscriptsand in 63% of the out-of-frame

tran-scripts.The VTI0-JP1,VT I0-J 1, and VT 10-J2 gene combina-tionsalsooccurredfrequentlyin both thein-frameand out-of-frame transcripts. Amongthe 232 clones sequenced, the JP2 gene segment was usedonly threetimes (weighed frequency 2.3%). In allthe donorsexcept one, Vy9-JPgene

rearrange-mentsweredominantamongthein-frametranscripts andwere rareamongtheout-of-frame transcripts.

Nodetectable transcription

of

the TCR VT]]gene in

adult

peripheral lymphocytes.

Asnotedearlier,the VyIV family-spe-cific probe didnothybridizetoany clones in theTCRy-specific

cDNA libraries. Conventional PCR reactions with a

_Cya

primerand aVyIV family-specific primeralso failedto

am-plify any TCRy gene product. To validate theability of the chosen

VyIV-specific

oligonucleotidetoprimethe VT11gene andtoverifythe presenceofarearranged VT11 gene in PBMC,

genomic DNA from several donors was amplified with the

VyIV

primerand the J 1 ,J2-specific primer (J 12). In each case, PCR yieldedthe expected 230-bpproduct, under conditions

where no detectable products were amplified in cDNA from PBMCusingthe sameVTIV primer(results not shown).

Col-lectively,these results indicate that TCR

VT

11transcription is

minimal in human lymphocytes that have rearranged TCR

V-yI I genes.

Thesourceofnonfunctionaltranscripts. On the basis of the

VTy

genefamily frequencies in the total transcripts from several donors and the sequence data, weestimatedthat 28-54% ofthe

transcriptswerenonfunctionalbecauseof frame shifts or

intro-duced terminationcodons. To discernthesourceof the

non-functional

TCRy

gene

transcripts,

PBMCs from one donor were sorted into

ca3

T cell-enriched and

_y6

T cell-enriched populations.RNAwasextractedfromeachpopulationorfrom totallymphocytesandTCRytranscriptswereamplifiedby

an-chored PCR. After constructing

TCRy

libraries, randomly

picked clonesweresequencedandexamined fortheir

potential

functionality.The resultsshowed that unfractionated

lympho-cyte population had 73% potentially functional TCRT

tran-scripts compared with27% inthe a: Tcell-enriched

popula-tion and 90% in the

_yb

Tcell-enriched population. Taken

to-gether, these data imply that most nonfunctional TCRy

transcripts

in

peripheral

blood

lymphocytes

comefrom a:T

cells.

Discussion

These experiments show that the expressed

potentially

func-tional

TCRT

generepertoire in adultperipheral lymphocytes derives fromalimitedsetof

V'y-Jy

combinations.

Only

11 of

40 potential

Vy-Jy

rearrangements were detected at a

fre-quencyof> 3%. Unrelatedsubjectshad thesamebasicpattern

ofVy-J-y geneuse. This restricted usageofparticular V-y-Jy gene segments could be explained by three possible

mecha-nisms.Oneisthe presenceofpotent andcommon

antigens

in

theperiphery,whichcanstimulate andexpandTcellsbearing particular types of

TCRy

genes. Another is the preferential rearrangement ofVy segmentstoJysegmentsatthe

progeni-tor T cell level. The last mechanism is positive or negative

selectioninthethymic environment,which isknown toaffect V: gene usage in peripheralT cellsof mice (12-15).

Todelineatehow theregulationof V-y-Jy gene recombina-tion places limitsonthemature

_yb

Tcellrepertoire, transcripts

genes werecompared. The frequencies of out-of-frame

V'y-Jy

gene rearrangements cannot be controlled directly by antigen selection in thethymusortheperiphery.The results showed that asimilarly restricted set of

Vy-Jy-Cy

genes was used in both functional and nonfunctional

TCRy

gene rearrange-ments. Most ofthe V'y-J'y combinations that appeared fre-quently in the in-frametranscriptswerealsofoundin the out-of-frame transcripts. As an example, the TCR J2 gene, rearranged to severaldifferent

Vy

gene segments, was found frequently in bothfunctional and nonfunctional transcripts.

The reputedlocations ofthe V, J, and C gene segments and theirfrequencies of recombinationin bothfunctionaland

non-functional transcriptsareshownin Fig. 1. Theprevalence of

V,y 10 and J2might be explained by positional effects, because

the TCR

Vy

10 geneisthe most downstreamV-ygene (except

for Vy 11) and J2 isthe most downstream J-y gene. In this regard, Triebel etal. ( 16) showed that the unexpressed

TCRy

allelesof eightclonedy T-celllineshaddownstream

V'Y

genes

joinedtoupstream

Jy

segments.Onthebasis ofanalysis of four

neonatal andthree fetal thymocyteclones, Krangel and col-leagues (5) proposed an ordered rearrangement of

TCRy

genes. McVay et al.(6) alsodescribedregulated expressionof

TCR,y andTCRbgenes infetalthymusand gut lymphocytes. However, theTCRygenes studied bythese workers had mini-mal Nregion additions,as has been seenwith both TCR and

immunoglobulingenes thatrearrangeearly in ontogeny (

17-21 ). In contrast, both the functional and nonfunctional

TCRy

genes in matureperipheral blood lymphocytes displayed exten-sive junctional diversity (Table II). Although the adult and fetal

TCRy

repertoires are not equivalent, the transcripts in mature peripheral blood

-y5

Tcells still derived from a limited setof V-J combinations. Despite the fact that ybT cellclones expressing

Vy

10 gene product have not been reported, we found manyfunctionally rearranged

Vy

10genes in peripheral blood lymphocytes. The discrepancy is probably due to the currentlack of specific antibodiesagainstthe

Vy

10region.

TheTCR

Vy

11 geneis known to be potentially functional, but it has been unclear if it can express a

TCRy

gene product. Inourexperiments, no V'y 11 gene transcription in peripheral blood lymphocytes was detected using either conventional PCR or anchored PCR, although we could readily amplify rearranged

Vy

11 genesfromthe samespecimens.These results suggest that TCR

Vy

11 genes arerarelytranscribed in mature

peripheralblood lymphocytes, in accordwith resultsrecently reported bytwoothergroups(21, 22). McVayetal.(6)used conventional PCR to demonstrate V111 gene expression in

fetaltissues. However, the PCRprimerthattheyused for

Vy

11 geneamplification (termed GVIV)isveryhomologoustothe

abundantly transcribed

Vy

10 gene,sharing20 out of 22bases,

and these workers noted that expression of the

Vy

11 gene was alwaysaccompanied by expression of theVy 10 gene. Indeed, wefoundthat thecloned Vy 10 gene wasreadily amplifiedby

Potentially functionaltranscripts

~

25.3%%

116.6%

9.2%-~

8.9%

-~ l

8.2%-7.0% _I 6.6%

5.3%

vy2 vy3Vy4 V' V8 Vy9 Vylo Vyl n _JP ji cyl JP2 J2 Cy2

4.1%, 4.6% 5.8%X 9.7%

13.9%-17.8%1

L - - - -0.9%0 -

--Non-functionaltranscripts

Figure1.Genomicorganizationofpotentiallyfunctional Vy, Jy,andC-ygenesonchromosome7and therecombinationfrequencies.Theeight

mostfrequentVy-Jy combinations inpotentiallyfunctionaltranscriptsand innonfunctionaltranscriptsareshown.Thefrequencyof Vy9-JP

innonfunctional transcripts is shown for reference.

176 H.Kohsaka,P. P. Chen, A. Taniguchi, W. E. R. Ollier, and D. A. Carson

I

PCR usingthe

Cy-specific

primers withthe GVIVprimer of

McVay,but not with the

VyIV

primer chosen forthe present

studies (datanotshown).

Becauseantigenic stimulationcanpotentially expandeven rareTcells inapopulation, theVy-J-ypairs in thefunctional TCRy repertoirecould bedifferent, and moreorlessdiverse,

thanthose in the nonfunctional repertoire. However, thetwo

generepertoireswere remarkablysimilar. The major

discrep-ancywas in the V-y9-JP transcripts, thataccounted for only

0.9% of the out-of-frame

transcripts,

but 25.3% (onthe

aver-age) of the in-frametranscripts. This predominance of func-tional Vy9-JPtranscripts wasobservedin seven of

eight

do-nors.The dataindicatethatlymphocytesexpressing rearranged

V-y9-JP-Cy

1 genes werenotpreferentially generated

during

'y6 Tcelldevelopmentbutinsteadwereexpanded

postthymically

fromsmallnumbersofprecursors.Consistent with this conclu-sion is the occurrence oftwo identical V'9-JP

joining

se-quences in randomlychosen

transcripts

fromthesamedonor (TableII).Themonoclonal

antibody TiyA recognizes

a

V-y9-associated

epitope

andstains the

major population

of

periph-eral y6 T cells in mostadults

(23).

Triebeletal.

(24)

showed that all

TiyA+

Tcelllines transcribe

V-y9-JP-Cy

1 rearranged

genes. Parker and

colleagues (25)

discovered that

Vy9

cellsare

notpredominantatbirth but expandin thefirst10 yearsof life.

Inthis regard,Tcellswith

V'y9/V62

TCR have been shownto

react with staphylococcalenterotoxin A,withthe Daudiand Molt-4lymphoblastoidcell

lines,

andwith

mycobacterial

anti-gens(26-29).

Althoughit is stillcontroversial ifprogenitor T cells only attemptaand : rearrangements when y and6rearrangements fail toproduce functional transcripts, TCRy gene

rearrange-ments areoften foundina13Tcells(30, 31 ). Some

af

lympho-blastoidcell linesare known totranscribe TCRy genes(32). The resultsofourexperiments with enriched

af3

and hyb T cell

populations suggest that thenonfunctional TCR-y gene

tran-scripts in peripheral blood lymphocytes also derive mainly froma: Tcells.

It ispossiblethat somefunctionally rearranged

_TCRy

tran-scripts comefrom af Tcells and thatdifferent Tcell clones

transcribeV^y genesatdifferent rates. However, it isvery

un-likelythatthesetranscriptsstronglyinfluencedthe overall

V'y-Jy frequenciesin the entireTcellpopulation. Thus, we found that Vy9-JP and Vy2- orVy4-J2 expression occurred most

frequently(averageof25.3 and14.8%; respectively)in the pool

of potentially functional transcripts.The prevalenceofthe two rearrangements agrees with previous enumeration analyses

carriedoutwithmonoclonal antibodies(3, 4).

In summary,the random assortment of V and J gene seg-ments in theTCR y locus can produce 40 different renants. Among the 232 genes that were analyzed, only 11

combi-nationsappearedfrequentlyin both in-frame and out-of-frame

transcripts. Thesedata show thatbiased

_V7y-J-y

rearrangement is animportantforce in molding the matureTCRyrepertoire. (The nomenclature used for human

TCRy

gene segments is thatofLefrancetal.

[33]

and Forster et al.

[34].)

Acknowledgments

The authorsacknowledgethesecretarialassistance ofMs.NancyNoon

inthepreparation of this manuscript.

Thisworkwassupported in partby grants AR25443 andAR40770

from theNational Institutes of Health and byagrantfromthe

Ciba-GeigyCorporation (Suncrest, NJ).

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