T cell receptor delta gene rearrangements in acute lymphoblastic leukemia

T cell receptor delta gene rearrangements in

acute lymphoblastic leukemia.

J Hara, … , M Minden, E W Gelfand

J Clin Invest.

1988;

82(6)

:1974-1982.

https://doi.org/10.1172/JCI113817

.

Using a newly isolated cDNA clone encoding the TCR-delta gene and genomic probes, we

have analyzed T cell receptor (TCR) delta gene rearrangement in 19 patients with T cell

acute lymphoblastic leukemia (T-ALL) and 29 patients with B-precursor ALL. Five out of

seven CD3- T-ALL and 4 of 12 CD3+ T-ALL showed bi-allelic rearrangements of the

TCR-delta gene. In three CD3+ patients, a single allelic TCR-TCR-delta gene rearrangement was

observed with rearrangement of the TCR-alpha gene on the other allele. In five CD3+

patients with bi-allelic rearrangements of the TCR-alpha gene, the TCR-delta gene locus

was deleted. Transcription of the TCR-delta gene was also analyzed in six T-ALL. Five

patients expressed TCR-delta transcripts. Only one T-ALL, presumably derived from the

most immature T lineage cells, did not have delta transcripts, but expressed

TCR-gamma and 1.0-kb truncated TCR-beta transcripts. In B-precursor ALL, 20 patients (69%)

showed rearrangements of the TCR-delta gene. The frequency of TCR-delta gene

rearrangement was higher than TCR-alpha (59%), gamma (52%), or beta (31%) genes.

These findings suggest that TCR-alpha gene rearrangements may take place after

rearrangements of the TCR-delta gene with concomitant deletion of rearranged TCR-delta

genes in T cell differentiation. Among leukemic cells of B lineage, the TCR-delta gene is the

earliest rearranging TCR gene, followed by TCR-gamma and beta gene rearrangements.

Research Article

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T

Cell Receptor

5 Gene

Rearrangements in Acute Lymphoblastic Leukemia

Junichi Hara,*Stephen H.Benedict,**EricChampagne,' Yoshihiro

Takihara,'

Tak W.Mak,'MarkMinden,

and Erwin W.

Gelfand**

*Division

of

BasicSciences and the Sackler Foundation Laboratory, Department ofPediatrics, National Jewish Centerfor Immunology and Respiratory Medicine, Denver, Colorado 80206; tDivision ofImmunology/Rheumatology, Research Institute, The Hospitalfor SickChildren, Toronto, Ontario,Canada MSG1X8;and Departments ofMedical Biophysics and Medicine, the Ontario Cancer

Institute, Toronto, Ontario, CanadaM4X1K9

Abstract

Usinganewlyisolated cDNA clone

encoding

the

TCR-M

gene andgenomic probes,we haveanalyzedTcell receptor (TCR) a gene rearrangement in 19 patients withTcell acute lympho-blastic leukemia (T-ALL) and 29

patients

with B-precursor

ALL.FiveoutofsevenCD3-T-ALLand 4 of12

CD3'

T-ALL showedbi-allelicrearrangementsof the

TCR-N

gene.Inthree

CD3+patients,asingle allelic TCR-5gene rearrangement was

observed with rearrangementof theTCR-agene on theother allele. In five

CD3' patients

with bi-allelic rearrangements of the TCR-a gene, the TCR-5 gene locus was deleted. Tran-scription of the

TCR-5

genewas also

analyzed

in sixT-ALL.

Fivepatients expressed TCR-6

transcripts.

Only

oneT-ALL, presumably derived from the mostimmatureT

lineage

cells, didnot haveTCR-5transcripts,butexpressed

TCR-y

and 1.0-kb truncated

TCR-,j

transcripts. InB-precursorALL, 20 pa-tients

(69%)

showed rearrangements of the TCR-6 gene. The

frequency of TCR-6 gene rearrangement was

higher

than

TCR-a

(59%),

S (52%),orj (31%) genes. These

findings

sug-gest that TCR-a gene rearrangements may take

place

after rearrangements of the TCR-5 gene with concomitant deletion ofrearranged

TCR-6

genes in T cell

differentiation. Among

leukemic cells of B lineage, the

TCR-5

gene is the earliest rearranging TCR gene, followed

by

TCR-'y

and # gene

rear-rangements.

Introduction

Acute

lymphoblastic leukemias

(ALL)'

are

presumed

toresult from clonal

expansion of

normal

developing

cells

(1-3).

Mo-lecularanalyses

of

leukemic

cells have

offered

important

in-sights

into our

understanding of

the

developmental

processes

of

lymphoid

cells

(4-1 1).

We

previously

analyzed

Tcell recep-tor

(TCR)-a

gene

configuration

in T-ALL and

B-precursor

ALL

using genomic

joining region (Ja) probes

covering

theJa

Dr.Hava's present address is Department of Pediatrics, Osaka

Univer-sityHospital, Osaka, Japan.

Addressreprint requests to Dr. Erwin W. Gelfand, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, CO 80206.

Receivedforpublication 19 April 1988 and in revisedform 19 July

1988.

1. Abbreviationsused in thispaper:C,constantregion;J,joining re-gion; TCR,Tcell receptor;UT, untranslatedregion.

regionup to85 kb 5'toCa (12, 13).All T-ALLwitha mature

phenotype (CDI-, CD3+) and 55% of B-precursor ALL showedrearrangementsof the TCR-agene. However, TCR-a

gene rearrangements were not observed in the majority of CD3 T-ALL orinCD1 +, CD3+T-ALL(13). An alternative

TCR, in whicha

y/5

heterodimer instead of

a/#,

is associated with the CD3 polypeptides, has been described (14-17). Thy-mocytes expressing

TCR-y/S

appearearly in ontogeny and are replaced by cells expressing

TCR-a/fl.

The subpopulationof

cellsexpressing

TCR-y/5

is aminoroneinthepostnatalperiod (18, 19). Presently, little is known about the function of

TCR-y/6,

and it remains uncertain whether cells expressing

TCR-y/6

belongto the same lineage as T cells expressing

TCR-a/f3.

Recently, a novel constantregion (C) gene was found in the TCR-a gene locus75 kb, 5'toCa in mice (20). This gene is believedtoencode theTCR-6 chain(21-24). HumancDNA clones analogousto this murine TCR-6 gene have also been recently isolated (25, 26). The human Cb has been located between theTCR-avariable (V) region and the TCR-a J

re-gion, - 90kb 5' to Ca. The human TCR-6 region contains at

leasttwodefined J6

loci

(25).

In

this

study, wehaveanalyzed TCR-6geneconfiguration in 19 T-ALLand 29 B-precursor ALLto delineate the rela-tionship between TCR-6 and TCR-a gene rearrangements.

Analysis

of

TCR-6 gene rearrangements used newly defined TCR-6 cDNA and genomic probes. Transcription of the TCR-6gene wasalsoexamined in6 T-ALLpatients.

Methods

Cellsamples.Mononuclear cells wereobtained from bone marrowby Ficoll-Hypaque centrifugationatthetime ofdiagnosisandbefore initi-ation oftreatment.Thesamplesevaluatedcontained>90%malignant

cells.Reactivityofmalignantcellswithapanelof MAbwasassessedby indirectimmunofluorescence (3).Thediagnosisof T-ALLwasbased

ontheexpressionof theTcell-associatedantigens CDI, CD2,CD3,

CD4, CD5, CD7, and CD8. Basedon CDI andCD3 expression,

T-ALLwas divided into four groups

(Table I);

group I, CDI- and

CD3-(Pts. 1-4); groupII, CDI'andCD3- (Pts.5-7);groupIII,CDl+ and CD33 (Pts. 8-14);and groupIV,CDI- andCD3Y (Pts. 15-19).In

all cellsamples, TCR-#ory gene rearrangementsweredetectedusing

the

C,9

probe (YT350.8-kb EcoRV-Bg IIIcDNAfragment) (27)orthe

Jy probe(0.7-kb Hind III-Eco RI fragment provided by Dr. T. H.

Rabbitts) (28)aspreviously reported (10, 11).

Thediagnosisof B-precursorALL wasbasedontheexpressionof the B cell-associated antigens CDl9 and/or CD20 and the lack of surface Igand Tcellandmyeloid-associatedantigens (29). IgHgene rearrangementswereobserved in all B-precursorALLsamplesusing

theCMprobe (1.3-kb EcoRIfragmentprovided byDr. T.H.Rabbitts)

(30)aspreviouslydescribed(3). Results ofTCR-y and ,B gene

rear-rangementspreviously reportedin part(10,11)areshown in TableIV.

Southern blothybridization. High-molecular weightDNAwas

ex-tracted from mononuclear cellsanddigestedwithrestriction

endonu-J.Clin.Invest.

© The AmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/88/12/1974/09 $2.00

Jal J62 C8

Hk

_{1 kb}

HH IH H.T Hif J

MH6 H5

Hinc11

5UT J81

H3- JG I 75 kb from Ca

H3 AEG

H-l

100bp

C8

Figure 1. (Top) A

re-strictionmapof the

TCR-6 gene and the

ge-nomicprobesusedin thisstudy. E,H,and B

denoteEcoRI,Hind

III,and Bam HI restric-tionsites,respectively.

(Bottom) The cDNA probe(cTH2) usedin this study.

cleases. The digested DNA was electrophoresed through 0.8% agarose gels and transferred to nylon membranes. Blots were hybridized to probes labeled with 32P by the random primer method (31). The TCR-a gene probes used in this study included six genomic Ja probes, JaB, C, D, E (2.3-, 4.7-, 2.0- and 4.0-kb Eco RI fragments, respec-tively), JaF (1.5-kb Hind III fragment), and JaG (6.7-kb Bam HI fragment). These probes weredescribed previously (12, 13). The JaB is the closest to Ca and the JaG is the furthest upstream, - 80 kb, as

showninFig. 1. The H3 probe (0.9-kb EcoRIgenomic fragment)was

also used to analyze the configuration of the most upstream Jaregion. The TCR-6 cDNA probe (cTH2) used in this studyincluded the un-translated (UT) region 5' toJ61, J61, C6, and the UT region 3' to C6 (Fig. 1). In cases where rearrangements were observed, blots were re-hybridized with the C6 probe (0.5-kb Eco RI-Hinc II fragment of cTH2). The MH6 (4.5-kb Eco RI genomic fragment) and the H5 (1.5-kb Xba I genomic fragment) probes were also used to detect rear-rangements involving the J61 andJ62 loci, respectively.

Northern blothybridization.

Cytoplasmic

RNAwasextractedafter

lysiswith NP-40 in the presence of 10 mM vanadyl-ribonucleoside complexandremoval of nuclei from mononuclear cells(32). 10'tgof

RNAwasdenatured informamide, electrophoresedin 1%agarosegels containing

formaldehyde,

and transferredto_nylonmembranes. Blots

were hybridized and washed under

high stringency

conditions

(0.1XSSC and 0.2% SDSat

_{650C) (33).}

Theprobesused in thisstudy

were asfollows:

_TCR-'y,

HGPO3 1.4 kb cDNA(34); TCR-6, cTH2 cDNA(described above);

TCR-fl,

Cj3 (described above);andTCR-a, Ca 0.35 kb Sau 3a-Hind IIIfragmentof cDNApGA5 (35).

Results

Rearrangement

of

the TCR-a gene in T-ALL. Results of TCR-a generearrangementobserved

_using

the

JaB, JaC,

JaD,

JaE,

JaF,

and JaG

probes

were

reported

previously

(12, 13),

andaresummarizedinTableI.One

(Pt. 4)

of four_groupI,one

TableI. TCR-6andaGene Rearrangementin 19 Patientswith T-ALL*

TCR-6

Surface markers cDNA Proposed

Jb gene a-6gene

Patient Groupt CD4 CD8 TCR-all MH6 5'UT C6, H5 usage configuration

I _ - _{G R/D R/D G G/R 62/61** 6/6}

2 + 30 G

R/Rt0

Rtt/D

R/G G 61/61**

6/6

3 - - G R/R D G G 61/61 6/6

4 + + R/R D D D D D a/a

5 + 42 G R/D D R/G G/R 61/62 b/6

6 II + + G R/R D G G 61/61

6/6

7 _ + R/G R/D D R/D G 61/D

6/a

8 + - _{G R/R D G G 61/61 6/6}

9 - _{20 R/G R/D D G G 61/D}

_6/a

10 40 40 G R/D D G G/R 61/62 b/6

11 III 19 - _{R/R D D D D D ala}

12 10 + R/G Rtt/D D R/D G 61/D

6/a

13 29 + G R/R D G G 61/61 6/6

14 + + G R/R R/D G G 61/61**

6/6

15 - - R/R D D D D D a/a

16 27 - _{R/G R/D R/D G G 61**/D}

_6/a

17 IV 42 45 R/R D D D D D a/a

18 - - R/R D D D D D a/a

19 + - R/R D D D D D a/a

*_{Gdenotesgermline, R,rearranged, andD,deleted.} *_{I:CDI-, CD3-; II: CDI+, CD3-;III:CDI+,CD3Y;IV:CDI-,CD3'.}

denotes< 10%positive cells, andaplussign>50%positive cells; numbersarespecificpercentagesof positive cells. "l Results usingtheJaB,

C,D, E, F,and Gprobes(13). 'Resultsafter Bam HI and Hind III digestion. **Rearrangements of the region_{upstream to}the J6 I_gene.

44_{Identified bythe}H3 probe.

§ Aminus

sign

(Pt. 7) of three group II, three (Pts. 9, 11, and 12) ofseven groupIII,and all five (Pts. 15-19)group IVpatients had

rear-rangements

of

the TCR-agene. Infour of these

patients (Pts.

7, 9, 12, and 16),germline configurationwasretainedonthe other allele.

TCR-6 gene rearrangement in T-ALL. DNAsamples were

digested

with Eco

RI,

Barn

HI,

orHind III and

hybridized

with theMH6, H5,orcDNA

probes.

Rearrangementpatternsand representative Southern blotsare shown in TableI and

Figs.

2-4.The MH6probe revealeda 14-kb BamHIband andtwo

HindIIIbands(5 and 2.6 kb)

(Fig.

2).The14-kbBam HIband and the5-kb HindIIIband contained the J51 gene.

Hybridiza-tion with theH5

probe

yielded

a4-kb Eco RI band

containing

theJ62gene

(Fig. 3).

The Eco RI

fragment

identified

by

the MH6probe, and Barn HI and HindIII

fragments

identified

by

the H5 probe could not be used to demonstrate rearrange-ments, asthese

fragments

didnotcontain the Jb1 orJ62genes

(Fig. 1).

All 13

patients

with

germline

configuration

of theTCR-a

gene on at least one allele showed rearrangements of the TCR-6geneusing the MH6 probe and either BamHIorHind

III digestion

(Fig. 2). Bi-allelic

J61 rearrangements were ob-served in 6 (Pts.

2,

3, 6, 8, 13,

and

14)

of these 13

patients.

The

remaining

seven

patients (Pts.

1,

5,

7,

9, 10,

12,

and

16)

showed

single

allelicJ6 1 rearrangementswithout retention of

G 1 2 6 7 8

14kb-germline configuration

ofthe TCR-6 gene. When these seven

patientswereexamined forJ62rearrangement using the H5 probe,three patients (Pts. 1, 5, and 10)demonstrated single

allelic rearrangements after Eco RI digestion (Fig. 3). These

findingssuggest that these three patients had rearrangements to Jb1 on oneallele andto

J52

on the otherallele. In all six.

patients with TCR-agene rearrangement onboth alleles (Pts. 4, 11, 15,and 17-19), the MH6 and H5

J5

bands were deleted.

ThecDNA probe (cTH2) was used to analyzeTCR-6gene

configuration in more detail. AfterEco RIdigestion, a 6-kb

bandcorrespondingtothe

5'JT

and

J6

1,andthree bands (3, 1.6, and 1.4kb) correspondingto

CG

and the

3YUT

were

ob-served in

germline

DNA (Figs. 1 and 4). Bam HI digests yieldedtwogermlinebands, a 14-kb bandcontaining Jb 1 and

J62, anda 12-kb band containingall the

C5

sequences (Figs. 1

and 4). Thesequences of thecDNAprobe encodingtheJbI

gene were too small to hybridize to the Jb1 gene under the

high-stringency

washing conditionsusedin thisstudy. In cases

of

rearrangements to the Jb1 gene, therefore, Jb1 bands

ap-pearedtobe deleted because the 5UTregion recognizedby the cDNA probe

(Fig.

1) is deleted in these types of rearrange-ments.

After

Eco RI or Bam HIdigestion,nine patients with rear-rangements previously identified bythe

MH6

probe showed

deletion of J61 bands on bothallelesusing thecDNA probe,

G 9 13 16

14kb--*

Bam H

I

G 1 2 3 5 7 G 9 10 12

*,

__ 5.3kb- f

5.3kb- AP

2.7

kb

-2.7kb

-Figure2.Representativerearrangement patternsof theTCR-6geneinT-ALL.Thepatient numbersare

noted above each lane. Lane G shows thegermline control.(A)Bam HI and(B) HindIIIdigestswere

probed withthe MH6probe. Asterisks indicatethe

fragments also identified by theH3probe. (A)Bam HIdigestsyieldeda14-kbbandingermlineDNA.

(B) HindIIIdigestsyielded 5.3- and2.7-kbbandsin

germlineDNA.

A

B

G~:A

21 5 6 9 10 13 16

.a..

:

rl

i ;+

e.

Eco RI

Figure3.Rearrangement patterns of the TCR-6 gene in T-ALL after

EcoRIdigestion and hybridization withtheH5 probe. TheH5

probe demonstrateda4-kb band ingermlineDNA.FaintbandsoW

servedbelowtheH5bandsareduetocross-hybridization.

suggesting rearrangements to the Jb 1 gene. In four patients

(Pts. 1,2, 14, and 16), rearrangedbands notidentified by the C0 probe (datanotshown)wereobservedwithoutgermlineJ6 1

bands. These rearranged bands were also identified by the

cDNAprobe after HindIIIdigestion. Asexpected, rearranged

Bam HI and Hind III bands were visualized with the MH6 probe. Usageof

J5

genesis summarizedin TableI.

Inthreepatients(Pts. 5, 7, and 12),afterBam HIdigestion

A

G

1 2 5 12 13 16

the cDNA probe demonstrated rearranged fragments identi-fied bytheCA probe despite germline configurationof Eco RI CAfragments (Table I). Similar resultswereobtainedby Hind

III digestion; 6.3-kb Hind III CAfragments correspondingto the most3'region ofCA alsoshowedrearrangements (data not

shown).Hybridizationwith the H3probedemonstrated germ-line Eco RI bands, butafterBam HI and HindIII

digestion,

rearranged bands were observedin these threepatients (data

notshown). Becausesizesof rearrangedH3bands were

differ-entfromthoseidentified bythe CAprobe,therewas

disconti-nuitybetweenH3andCA genes.Furthermore, therearranged Bam HIand Hind III Jb1 bands in Pt. 2 and the rearranged Hind III Jb 1 band in Pt. 12 wereidentified bytheH3 probe suggesting recombination between J61 and theregion

identi-fiedby the H3probe.

TCR gene transcription in T-ALL. Results ofTCR-,y, f,

andagene

transcription

reported

previously (13)

are

summa-rizedinTable II.TCR-6genetranscriptionwasanalyzedinsix

patients using

the cDNAprobe. Northernblotsareshown in

Fig. 5. TheleukemicTcellline, PEER, with

TCR-,y/b

onthe cellsurface ( 17) expressed four sizesof

transcripts,

2.2, 2.0,

1.5,

and 1.3 kb.Of these,2.2- and1.5-kb

transcripts

were

predomi-nantly expressed and the2.0-and 1.3-kb

transcripts

were

visi-bleinalongerexposureofthe

experiment presented

in

Fig.

5. 2.2- and 1.5-kbtranscriptsarecomposed of

full-length

mRNA

containingVregions;ontheotherhand,2.0- and 1.3-kb

tran-scriptsareimmature andlack V

region

transcription (25).

The size differences between 2.2- and 1.5-kb

transcripts

and be-tween2.0-and 1.3-kb

transcripts

are most

likely

dueto

differ-ences in

polyadenylation

sites (25). One

patient

(Pt. 1), who onlyexpressedtheCD7

antigen

andtherefore presumably de-rived from immature T

lineage

cells, did not express the

TCR-6 gene. In this patient, neither TCR-a nor full-length

B

G 1 2 14

5

6

7 12 10

G8

J6(14.0)

-C8(1

2.0)

- _/

--ww _/-,

C6(3.0)-

__

_

_

*Mow~~~~~~~~~~~~~~~~~~~~~~~~~~~F.

C6(1

.6)

C6 1.45-4

--0

Eco R I

Figure4.Representativerearrangement patternsof the TCR-6gene

in T-ALL.(A)Eco RI and(B)Bam HIdigestswereprobed withthe cDNAprobe (cTH2). Rearrangedbandsareindicatedbyarrows on

the blots.(A)Eco RIdigests yieldeda6.0-kbbandcorrespondingto

C0

Bam HI

the5'UT andJb1 and 3bands, 3.0, 1.6,and 1.4 kbcorrespondingto theCb. (B)Bam HIdigests yieldeda 14-kb bandcorrespondingto

the5S JTandJb1 anda 12.0-kb bandcorrespondingtotheCb.

Rear-rangedbandscorrespondingtoC0 areindicatedbybarsonthe blot.

fI-Table I. Transcription of TCR Genes in11 Patientswith T-ALL

Transcriptiont

a6z ,B a

Rearrangement

Patient Group* 6, a 2.2 2.0 1.5 1.3 1.7 1.3 1.0 1.6 1.3

1 a/6 - - - - _{+ +}

2 I 3/6 +(wk) + +(wk) + + + - - _

4 a/a + +

9 5/a +(wk) + +(wk) + + + + +

-10 I/I + +(wk) + +(wk) + - + +(wk) +(wk)

11 a/a - + + +

12 /'a - +(wk) +(wk) + + + + +(wk) +(wk)

16 6/a +(wk) + +(wk) + + + - +

-17 V++a+ +

18

V/a

+ + + + +

19 a/a + +

*_I:

_CD1-,

_{CD3-; III:CDI+, CD3+;IV:}

CDl-,

_{CD3+. $Size inkilobases.+denotes readilydetectable expression; +(wk), weak expression;-,}

expressionwas notdetected.

1.3-kbTCR-f transcriptswereobserved, but the

TCR-'y

gene was expressed. Theremaining five patients(Pts. 2, 9, 10, 12,

and 16) expressed the TCR-6 gene along with

_TCR-7

tran-scripts. The

transcriptional

intensity

patternsinfour

patients

(Pts. 2, 9, 12, and 16) were different from those in PEER.

Levelsof 2.2- and 1.5-kb transcriptswere lowcomparedwith

2.0- and 1.3-kbtranscripts. Onlyonepatient (Pt. 10)strongly

expressed 2.2- and 1.5-kb TCR-6 transcripts and showed a

similar

transcriptional intensity

patterntothatobserved with PEERcells. Inthis patient, TCR-,yandonly 1.0-kbtruncated

TCR-, transcriptsweredetected. In

addition,

weakexpression of the TCR-a gene was demonstrated. It is

likely

that these

TCR-a transcripts may be derived fromthe TCR-a gene in germline

configuration.

TCR-agene rearrangement inB-precursorALL. Resultsof

TCR-ageneconfiguration in B-precursorALLobtained

using

theJa probes will be

published

elsewhere (36a). 17 patients showed rearrangements of the TCR-ct gene. In five patients (Pts. 44-48), TCR-a gene rearrangements were

bi-allelic

and in 12patients(Pts.32-43), the rearrangementwas on asingle allele(Table III).

PEER

2

9

10

16

12

1

2.2-

1

2.0

-1.3

-A.

Figure5.Transcriptionof the TCR-6gene.Sizes of transcriptsare

indicatedontheleft side ofthe blot.

TCR-6generearrangementinB-precursor ALL. Resultsin

29 B-precursor ALL obtained using the cDNA and MH6 probes are summarized in Tables III and IV. Representative Southern blots are shown in Fig. 6.TCR-6gene rearrangement wasobserved in 20 patients (Pts. 23-31 and 33-43). 11 (Pts.

33-43) ofthese 20 patients also had rearrangements of the TCR-a gene on the other allele. In five patients (Pts. 44-48),

the TCR-6 locuswasdeletedon both alleles and TCR-a gene was rearranged. In contrast to findings in the majority of T-ALL, all B-precursor ALL with TCR-6 gene rearrangement showed an unusual rearrangement pattern. Rearranged J61 bands were observed when Eco RI or Bam HI digestswere

probed with thecDNAprobe. This indicated that intervening

sequences betweenD6andJb1 genes werepreserved in these rearrangements and suggests that unlike the case with Ig genes, D to J rearrangement may not be the first event in TCR-6

rearrangement.

Asexpected,rearrangedBam HI bandswerealso identified

bythe MH6 probe(datanotshown). As shown in Fig. 6, all rearrangements, except for those observed in patients with three rearrangements, fell into only three groups. The H5 probe failed to demonstrate rearrangements in any patients withB-precursor ALL(datanotshown).Two

patients

(Pts.25 and 27) with germline configuration of the TCR-a gene on bothalleles hadonlyasingleMH6rearrangedband. It is

possi-ble that rearranged bands onboth alleles were the same size andoverlaideachother,

preventing

discrimination. Basedon

the

configuration

of theTCR-6anda genes, 29patientswith B-precursor ALL were divided into fivegroups, as shown in Tables III and IV. Only three patients (group A) showed

germline configuration of both theTCR-6andagenes. Nine

patients (group B)had rearrangementsoftheTCR-6 gene with

germline

configuration

of the TCR-a gene. In one patient

(group C), TCR-a gene rearrangement (JaG) was observed with asinglegermline allele of the TCR-6 gene. Theremaining

TableIII. TCR-a and5GeneRearrangement in 29 Patients with B-precursor ALL

TCR-6

a-6gene

Pt. Group* TCR-at MH6 cDNA(5'UT) configuration

20 A G§ G G G

21 G G G G

22 G G G G

23 B G R/G R/G b/G 24 G R/G R/G 6/G

25 G R/D R/D 6/6 26 G R/R R/R 5/6

27 G R/D R/D 5/6

28 G R/R/R R/R/R b/5/6

29 G ND RIG 5/G

30 G R/R R/R 5/6

31 G R/R R/R 5/6

32 C R/G D/G D/G a/G

33 D G/R ND R/D 5/a

34 G/R R/D R/D 5/a 35 G/R R/D R/D 5/a

36 G/R R/D R/D 5/a

37 G/R R/D R/D 5/a

38 G/R R/D R/D 5/a

39 G/R R/D R/D 5/a

40 G/R R/D R/D 5/a

41 G/R R/D R/D 5/a

42 G/R R/D R/D 5/a

43 G/R R/D R/D 5/a

44 E R D D a/a

45 R D D a/a

46 R D D a/a

47 R D D a/a

48 R D D a/a

* See text for description of group designations. Resultsusing the JaB, C, D, E, F, and G probes (36a). § G denotes

germline, R,

rearranged, andD,deleted.

TCR-6gene andin5patients(groupE) TCR-6wasdeleted on bothalleles.

Discussion

Inthisstudy, wehaveanalyzed TCR-6gene

configuration

in

T-ALL and B-precursor ALL usinga newly isolated human

TCR-6cDNAprobe andvarious

genomic

probes. Among 19 T-ALL patients, 9 patients demonstrated rearrangements of

theTCR-6gene onbothalleles and4

patients

wererearranged

on asingleallelewith rearrangementsofthe TCR-a geneon

the other allele. The remaining six patients had biallelic TCR-agene rearrangements and the TCR-6 gene was deleted.

Transcription ofthe TCR-6 gene was detected in five of six patients analyzed. B-precursor ALL also showed ahigh

fre-quencyofTCR-6 gene rearrangements. 20of29 B-precursor

ALLpatientshadrearrangements of the TCR-6 gene and 11 of these 20patients had TCR-a gene rearrangements on the other allele. Of the remaining nine patients, bi-allelic TCR-a gene rearrangements wereobservedinfivepatientsand onepatient

had asingle allelicTCR-a gene rearrangement with germline

configurationofthe TCR-6 and agenes. Only three patients

didnotshow rearrangement of the TCR-6 oragenes. Based on theexpression oftheCDl and CD3 antigens,the

patients with T-ALL studied here were divided into 4 sub-groups;group I, CD1- and CD3-; group II, CDl+ and CD3-; group III, CDl+ and CD3+; and group IV, CD1- and CD3+.

Thesegroups arepostulatedtorepresentprogressivestages of Tcelldifferentiation.Three of four group I patients (75%) and two of threegroup IIpatients (67%) had bi-allelic rearrange-ments of the TCR-6 gene. One of thegroup I patients had bi-allelicTCR-a gene rearrangements, and one of the group II

patients showed TCR-6 and TCR-agene rearrangements on

each allele. Among theCD3+ T-ALLpatients, four of seven group IIIpatients (71%) showed bi-allelic rearrangements of the TCR-6 gene and two patients showed TCR-6

rearrange-TableIV. SummaryofTCRGene Rearrangementin 29 Patients

withB-precursor ALL

Tcellreceptor genes

Pt. Group a a y

20 A G* G G G

21 G G G G

22 G G G G

23 B R/G G G G

24 R/G G G G

25 R G G G

26 R G G G

27 R G R/G G

28 R/R/R G R/G G

29 R/G G R/G G

30 R G R G

31 R G R/G G

32 C D/G R/G G G 33 D R/D G/R G G 34 R/D G/R G G 35 R/D G/R G G 36 R/D G/R G G

37 R/D G/R G G 38 R/D G/R R/G G 39 R/D G/R R/G G 40 R/D G/R R R/G

41 R/D G/R R R

42 R/D G/R R R/D

43 R/D G/R R R/G

44 E D R G R/D

45 D R R R

46 D R R R/G

47 D R R R/G

48 D R R R/G

A

G 27 30 31

G

26

28 27

30

J6(14.0)

_

_

2.0):- NOW.

.

_

C6(1.6)

-

C6(1.4)-0A = ~ k' t

Eco

RI

ment on one allele and TCR-a gene rearrangement on the

other allele.Bi-allelicrearrangementsof the TCR-a gene were

observed intheremaining patient. In contrast to results with group III, all group IV patients had TCR-a gene rearrange-mentandonlyonepatientshowed TCR-6 gene rearrangement

in addition to TCR-a gene rearrangement. Based on these

findings, itseemsthatatearly stagesofdifferentiation,TCR-6 gene rearrangement may initially occur and during further

differentiation,

rearrangements of the TCR-agene may take place with concomitant deletion ofa preexisting VDJ

CO

complex. It is possible that successful rearrangement of the

TCR-6 genetogetherwith functional TCR-y gene

rearrange-mentmayproceeddirectlytodifferentiate intothe T cell

sub-set

expressing

TCR-'y/b

onthe cellsurface.If,however, TCR-6

orTCR-' rearrangements prove nonfunctional, this may

re-sult in further rearrangement ofthe TCR-a gene locus and

these cells would

differentiate

intoTcellsexpressing

TCR-a/f3.

Analogousfindings have been observedwith Ig genes where

functional

rearrangementsof the Ig-Kgenemayinhibit activa-tionof the Ig-Xgenelocus; failure of successfulrearrangement

oftheIg-Kgeneinitiates Ig-Xgene rearrangementwith deletion of the Ig-K genelocus (36).

Alternatively,

thesetwo setsofT cells mayderive fromseparate

lineages.

Interestingly, most group III

patients

(CD1+ and CD3+) showedrearrangementof the TCR-6genewith

germline

con-figuration oftheTCR-agene. It has beenthoughtthat

expres-sion oftheCD3 complexonthe cellsurfacemayrequirethe presenceofthe

TCR-a/,3

heterodimer (37-40).Ifoneassumes that the

TCR-y/6

heterodimermaysubstitutefor

TCR-a/f

in CD3

antigen expression,

the

CD3+

cellswithbi-allelic TCR-6

gene rearrangement observed in this study should have the

TCR-'y/6

inassociationwith the CD3 complex.The

majority

of

freshly

isolated CD3+ double

negative

(CD4- and CD8-) thymocytes expressedtheCD1

antigen

andafterculture with

IL2, CD1 disappeared from the surface of cells

expressing

TCR-'y/b

(15). Itis,therefore,likelythatalargeproportionof cellswith TCR-y/b may express CD1 in vivo. In contrast to

group III

patients,

all five group IV

patients

had

rearrange-ments ofthe TCR-a gene. It would be

interesting

to know

Bam HI

Figure 6. Representative rearrangementpatternsof the TCR-6genein

B-pre-cursorALL.(A)EcoRI

and (B)Bam HIdigests were probed with the cDNAprobe(cTH2).

Rearrangedbandsare in-dicated byarrows onthe blots.

whether those

y/-bearing

cells,belongingtogroup III, differ-entiate into group IV cells withreplacement of

TCR--y/b

by

TCR-a/f3.

AsshowninTableI, 15ofthe 22 total rearrangementsof

theTCR-6 gene in T-ALL used the JO 1 gene. In contrast,useof the J02 genewasobservedonlyinthree rearrangements. The

remaining four rearrangements in T-ALLand all rearrange-mentsobserved inB-precursor ALL wereincompatiblewith

ordinary DJO or VDJO rearrangements. In these rearrange-ments,interveningsequencesbetweenDb andJO1 geneswere

preserved,suggesting that VDO rearrangements

might

insome casesprecede rearrangement of DbtoJO. Sequence data(26)

suggestthepresence ofaD gene atthe 5'terminus of the Jb1 locus(Hind III-Eco RIfragment)and that noJO genes other than the JO 1 gene encodedbythecDNAprobeexist within this locus. Ittherefore seemsthat rearranged bands identified by

the cDNAprobedidnotresultfrom

ordinary

DJO1 orVDJO1

recombinations.Thelikely explanation of this phenomenonis

aVDO rearrangement withpreservationoftheintervening

se-quences between Db

and

J6O genes.

Alternatively,

some

un-knownmechanism

regulating

theassembly oftheTCR-6gene

mighthaveresultedin these rearrangements.Thistypeof

rear-rangementhas been demonstratedfor the TCR-6genein fetal

mousethymocytes

(41),

andseemsto occurfirstinthese cells

atleastasfrequentlyasDtoJ rearrangements.

Four patients with T-ALL(Pts. 2, 5, 7, and

12)

showed curious patterns of rearrangementwhichweredifficultto

ex-plain by

ordinary

recombinational models. In these

patients,

discontinuity

betweenthe

rearranged

CO and the

region just

5'

toH3wasobserved. InPt.

2,

recombinationbetweenJO1 and

the

region just

5' to H3 was demonstrated with retention of

germline

configuration

of the JO2 and CO genes. Patient 12 also showed recombination between thesetwoloci after Hind III

digestion.

Although

precise

characterization of the other

re-combination siteswas not

completed,

these

findings

suggest thataninversion of the locus that included theJOandCOgenes had occurred. Recently,

repetitive

elements located around

this

region analogous

to switch

regions

in the

IgH

constant

regionhave been described

(42).

These elementsremainedin G 26 28

B

germline configuration in T cells expressing

TCR-,y/b

and were deletedfromTcellsexpressingTCR-a/f. Rearrangement

of these elements hasonly been observed in polyclonal thy-mocytes, including putative cellsshowing transient character-istics between immature T cells, which express

TCR-y/6

and mature T cells, which express TCR-a/fl (42). Although the

location of these repetitive elements in the genome is un-known, it is possiblethat leukemic cells from these patients

with rearrangementsofthe regionjust5' to H3 may be arrested

at a normally transient stage between stages expressing

TCR-y/6

and cellsexpressing TCR-a/f3,and recombinational

eventsobserved inthesepatients may reflectatransition

be-tweenthese twostages. Wearecurrently attemptingto

delin-eate the recombinational eventobserved here by sequencing

the potential recombination sites. Alternatively, since the Ja

region, andprobablytheTCR-6 gene locusarefrequently in-volved in translocations in T cellneoplasia(43-45),

chromo-somal abnormalities involving chromosome 14 may have causedthese abnormalitiesatthisgene locus.Indeed,Pt. 5had

atranslocation of t(10; 14) (q 24;q 11).Recentdatafrom Isobe

etal. support thispossibility (46).

To assess expression ofthe TCR-6 gene, Northern blot analyseswere performed usingthe cDNAprobe. There were

foursizes of TCR-6 transcripts;thesewere2.2, 2.0, 1.5,and 1.3 kb. The2.2- and 1.5-kbspecies have been showntobe func-tional transcripts containing VDJ sequences and these were

predominantly expressed in the leukemic T cell line PEER

expressing TCR-y/6

on the surface(25). A similar pattern of TCR-6transcriptswasobserved inPt. 10withbi-allelicTCR-5

rearrangement. In this patient,

TCR-'y

transcripts were also detected but 1.3-kb, full-length TCR-f3 transcripts were not

present (Table II). Itis likely thatthecells from Pt. 10 have

TCR-y/5

in association with the cell surface CD3 complex. Conversely, 2.0- and/or 1.3-kb

transcripts lacking

V

regions

(25)wereabundant,compared with2.2-and1.5-kbtranscripts

infourpatients(Pts.2,9, 12,and16). Of these,the threeCD3+

T-ALL(Pts.9, 12,and 16)also hadfull-length, 1.6-kb TCR-a transcriptsand

full-length,

1.3-kb TCR-f transcripts,

suggest-ing expression ofthe

TCR-a/3

complex on the cell surface.

Onepatient (Pt. 1), presumably

representing

themost

imma-ture T cells, failed to express TCR-6 transcripts, despite

ex-pression of TCR-yand 1.0-kbimmature

TCR-3

transcripts. Althoughsurprising, it is possiblethatsomeTCR-6

transcrip-tionoccurredearlier,tofacilitate TCR-6rearrangement. This

TCR-6transcription may have been abortive and theTCR-'y

transcription

nowobserved reflectsasubsequentstep in devel-opment. Thus

transcription

of the

fully

rearranged TCR-6

gene may bepreceded by

transcription

of rearranged TCR-,y

and TCR-6transcripts might beacriticaleventfor

TCR-,y/6

expression

on the cell surfaceina situation analogoustothe

relationshipbetween theTCR-,Bandagenes.

Among 29 patients with B-precursor ALL, 20 patients (69%) showed rearrangements ofthe TCR-6 gene. The fre-quency ofTCR-6gene rearrangement was higher than TCR-a

(59%),

y (52%), and ,B (31%) genes. Nine patients showed

TCR-6 gene rearrangement without rearrangements of the

TCR--y orf genes and

TCR-3

gene rearrangement

accompa-nied TCR-,y gene rearrangement in all but one patient.

Al-though,asdiscussedabove, the nature of these rearrangements of TCR-6 is still obscure, it appears that at least in leukemic

cells of B

lineage,

TCR-6 gene rearrangement is the earliest

eventamong rearrangements of the TCR genes. Itis also possi-ble that rearrangementsofthe TCR-6 and TCR--y genes may

be followed by rearrangements of the TCR-a and

_TCR-#

genes, respectively. TCR-a and 6 rearrangement may in turn

be independently regulated by some protein products of rear-ranged TCR-6 and

ey

genes. To conclude whether the hierarchy derived from this study of B lineage cells reflects the order of TCR gene rearrangements in T lineage cells, it will be neces-sary to analyze a greater number of immature cell samples.

Acknowledgments

We are indebted to the members of the Division of

Hematology/On-cology, TheHospital for SickChildren, forproviding patient

speci-mens. Dr. T. H. Rabbitts and Dr. J. Kappler kindly provided the DNA probes.

Thiswork was supported in part by theNationalCancerInstitute, the National Institutes of Health and IM-510 from the American

Cancer Society. Dr. Harawas arecipientofaTerryFoxFellowship

award; Dr.Gelfand isaScholarof the Raymond and Beverly Sackler Foundation.

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