T cell receptor alpha , beta , and gamma genes in T cell and pre B cell acute lymphoblastic leukemia

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T cell receptor alpha-, beta-, and gamma-genes

in T cell and pre-B cell acute lymphoblastic

leukemia.

C A Felix, … , P Goldman, S J Korsmeyer

J Clin Invest.

1987;

80(2)

:545-556.

https://doi.org/10.1172/JCI113103

.

We examined alpha-, beta-, and gamma-T cell receptor (TCR) gene activation within acute

lymphoblastic leukemias (ALLs) that represent early stages of B and T cell development.

We wished to determine if TCR rearrangement and expression was lineage restricted,

showed any developmental hierarchy, or could identify new subsets of T cells.

Rearrangement of gamma and beta TCR genes occurred early in development but in no set

order, and most ALLs (22/26) were of sufficient maturity to have rearranged both genes.

T-ALLs preferentially rearranged C gamma 2 versus the C gamma 1 complex; no preference

within the beta locus was apparent. Once rearranged, the beta TCR continued to be

expressed (11/13), whereas the gamma TCR was rarely expressed (3/14). The alpha TCR

was expressed only in more mature T-ALLs (8/14) that usually displayed T3. The 3A-1 T

cell associated antigen appeared earliest in development followed by T11 and T3. Within

pre-B cell ALL a higher incidence of lineage spillover was noted for gamma TCR

rearrangements (8/17) than for beta rearrangements (3/17). This also contrasts with the only

occasional rearrangement of immunoglobulin (Ig) heavy chains (3/25) in T-ALL. However, in

pre-B ALL the pattern of gamma TCR usage was distinct from that of T cells, with the C

gamma 1 complex utilized more frequently. Almost all ALLs could be classified as pre-B or

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T

Cell Receptor

a-,

fl-,

and

'y-Genes

in T

Cell

and

Pre-B Cell Acute Lymphoblastic Leukemia

Carolyn A. Felix, John J. Wright, David G. Poplack, Gregory H.Reaman,DianeCole, Paula Goldman, and Stanley J. Korsmeyer

Metabolism and PediatricBranches,National Cancer Institute, Bethesda, Maryland 20892; Department ofHematology-Oncology,

Childrens Hospital National Medical Center, George Washington UniversitySchoolofMedicine, Washington,DC20010; Departments ofMedicine, Microbiology, andImmunology, Howard Hughes Medical Institute,

Washington University School ofMedicine, St. Louis, Missouri 63110

Abstract

We examineda-,,(-,and'y-Tcell receptor(TCR)geneactivation

within acute lymphoblastic leukemias (ALLs) that represent

earlystagesof Band T celldevelopment.We wishedtodetermine

ifTCR rearrangement and expression was lineage restricted,

showedanydevelopmental hierarchy,orcouldidentifynew sub-setsof T cells.Rearrangementof'yand,( TCRgenesoccurred

earlyindevelopmentbut innosetorder,andmostT-ALLs(22/ 26)wereof sufficient maturity tohaverearranged bothgenes. T-ALLs preferentially rearranged

C.y2

versusthe

Cj1

complex; nopreference within

thef(

locuswasapparent.Once rearranged,

the TCR continued to beexpressed (11/13), whereas the y

TCRwasrarely expressed (3/14). TheaTCRwas expressed onlyinmorematureT-ALLs(8/14)thatusuallydisplayedT3. The3A-1 Tcell associated antigen appeared earliest in

devel-opmentfollowedby T111 andT3. Withinpre-Bcell ALLahigher incidence of lineage spillover wasnoted for y TCR

rearrange-ments(8/17) thanfor rearrangements (3/17).Thisalso

con-trastswiththe only occasionalrearrangementof immunoglobulin (Ig)heavy chains (3/25)inT-ALL. However,inpre-BALLthe

pattern ofyTCRusagewasdistinct from that of Tcells,with

the

C,1

complexutilizedmorefrequently.Almost all ALLs could beclassifiedaspre-BorT cell in typeby combining Igand TCR

geneswithmonoclonal antibodies recognizing surfaceantigens, although examples oflineage duality werenoted.Unique

sub-populations of cells were discovered including twogenetically

uncommitted ALLs that failed to rearrangeeither Ig orTCR loci. Moreover, two T lymphoblasts were identified that pos-sessedtheT3moleculebut failedtoexpressaplus,( TCRgenes.

These T-ALLsmayrepresent afortuitous transformation of T cellsubsets with alternative T3-Ti complexes.

Introduction

Theantigen-specificTcellreceptorgenesprovidethefirst

uni-versally applicable markers ofT cell clonality. The

character-Address reprintrequests toDr. Korsmeyer, Department of Medicine and Microbiology/Immunology, Washington University School of

Medicine, 660 S. EuclidAve.,St. Louis,MO 63110.

Receivedfor publication 21 August 1986 andinrevisedform13

Feb-ruary1987.

ization ofthe T cell receptor

(TCR)'

lociin

lymphoid neoplasms

should

improve

our understanding of lineage

commitment,

clonality, stage of differentiation, and T cell associated chro-mosomal translocations. In this study we examined

rearrange-mentand expression of TCR genes within acute lymphoblastic leukemias of both T cell and pre-B cell type. We wished to de-termine whether the activation of a,

fl,

and -yTCR geneswas

lineage restricted and whether this was useful in placing early lymphoid progenitors into T cell versus B cell lineages.

Moreover,

wesought to determine if there was a hierarchy of a, (3, and

oy

TCRgeneutilization, if it correlated with T cell surface antigen expression, and whether this information could pinpoint the

stateofdifferentiation of individual T cell ALLs.

Wepreviously demonstrated that the non-T ALLs that lacked T cell surface antigens were adevelopmental series of B cell precursors (1-3). All of these pre-B ALLs had rearranged im-munoglobulin (Ig) heavy (H) chain genes and- 40% had

pro-gressedtothe later developmental step of rearranging theirKor

X light (L) chain genes. Coordinate with this

hierarchy

ofIg gene rearrangements wasanorderedexpression of B cell associated surfaceantigens in which B4 and HLA-DRwerepresentonthe mostimmature cells, the commonacutelymphoblastic leukemia antigen (CALLA) followed, and Bl was present on the most maturecells.Approximately 20% of ALLs bore T cell associated surface antigens (4, 5), uniformly retained germline Ig L-chain andusually (90%) germline Ig H chain genes (2), andwere

con-sequently

felttorepresent T cell

lineage

ALLs. A number of T cellassociated surface antigens have been identified, however

none ofthese are

malignancy

restricted nordo

they

serve as

clonal markers.

Consequently,

we

investigated

the TCR genes in T-ALL to determine ifan

analogous

cascade

of

rearrange-ments occursinpre-Tasit does in pre-B development. The classic antigen specific T cell receptor isaTi heterodimer oftheaplus 13 TCR chains that is

complexed

intramembranously toform the T3/Ti molecular complex (6). T3-Ti

expression

is noted only in the most mature

thymocytes

and

peripheral

T cells(7, 8). The human I TCR gene is locatedonchromosome 7atband q35 (9) and is

comprised

oftwo constant

(Cal)

segments, 13joining (Jo) segments, two or more

diversity

(Do)

segments, and -100 variable

(VO)

gene segments(10-12). Rearrangements of this locus are

readily

detected and intermediate

(D/J)

1.0 kilobase (kb) and complete (V/D/J) 1.3 kb

transcripts

can be discerned (13-18). Thea TCR locus is foundat

14qI

1 and is

1.Abbreviations usedinthis paper: CALLA,common acutae lympho-blastic leukemiaantigen;EBV,EpsteinBarrvirus;sRBC, sheep red blood cell;TCR, Tcell receptor,tdt, terminal deoxynucleotidyl transferase;

WBC, white blood cells.

J.Clin. Invest.

©TheAmericanSociety for Clinical Investigation,Inc.

0021-9738/87/08/0545/12

$2.00

(3)

particularly prone to interchromosomal recombination in T cell ALLs (19-22). Itconsists ofone

C,

region, an exceptionallylong stretch of Ja segments that may contain 50-100 J regions spread over morethan70 kb. No diversity segments are known to exist in the a TCR locus, but there are probably 40 or more

V.

regions (23-25). While the markedlylong stretch ofJas makes it currently rather impractical to search for rearrangements in all cells, RNA transcripts of complete recombinations (1.6 kb) and truncated forms (1.3 kb) can be assessed (23). In addition to the a- and (3-chain that

contribute

to theTi heterodimer, at least one other TCR gene, yis known to exist (26-29). This rearranging deter-minant is found on chromosome 7 at band p1 5 (30). In humans twoC.s are present, together with three or four Jzs, and perhaps inthe range of 10

V,

segments. Despite having a similar

orga-nization

to the other TCR genes the precise function of this locusis uncertain (31, 32). Because of this, we wished to assess its rearrangement and expression in T-ALLs to gain potential insights into its role in T cell development.

Developmental studies in

the mouse suggested a hierarchy of TCR gene

activation

in which yTCR expression might pre-cede the(3 TCR. y TCR RNA decreased dramatically during

intrathymic maturation, while

(3RNAremained constant, and a RNA

increased

(6, 7, 33-36). We sought to determine whether

the T-ALLs represented

a

population of

cells early enough

in

pre-T development to reveal any ordered usage of TCR genes

in

man. Moreover

by

examining

T3 andother cell

surface

an-tigens together

with a,

(, and

ywe also

sought

potentially

new

subpopulations of

T

cells. The

comparison of

Ig

and

TCR genes

in

pre-B and T cell ALLs was

performed

to assessthe

lineage

restriction of these developmental

events and to determine

if

any

dual-lineage

or

clearly

uncommitted

lymphoid progenitor

cells existed.

Methods

ALLsexamined.Leukemic cells were obtainedby

Ficoll-Hypaque

gra-dientcentrifugationof bonemarrow

aspirates

or

leukapheresis

obtained

lymphocyte fractions from peripheral blood ofpatients with ALLat

eitherdiagnosisorrelapse.Sampleswereavailablefrom 23

patients

cat-egorizedas Tcell ALL and 17patients classifiedaspre-BcellALL.In

addition, threeestablished T-ALLcell lines

(HSB, CEM,

and RPMI

8402)andfourpre-Bcelllines

(RS, Nalm-6, Nalm-l,

and

Reh)

were

alsoexamined (2,37, 38).Also studiedwere

examples

ofmore mature

BcellneoplasmsincludingaBcell chronic

lymphocytic leukemia,

five Burkittlymphomacelllines

(IARC

LY47,IARC

LY67,

IARCJI,IARC LY66,Raji),andsixBcelllymphomacelllines(SU-DHL-4,

SU-DHL-6,SU-DHL-7,

SU-DHL-8,

Balm-l, Balm-4) (39-41). Moreover,

14clonal

EpsteinBarr Virus transformedBcell lineswereutilized.The Hut 102 cell linewasutilizedas anexampleofa matureTcell(42),while K562 representedan

erythroleukemia

(43), HL-60apromyelocyte(44),and SU-DHL-Iahistiocyte (40).

ALLswereinitiallyclassifiedas Tcell type ifthey possessedTcell associatedantigens (3A-1,andattimesT

11

and

T3),

lackedBcell

as-sociatedantigens (B4and

Bl),

and retained

germline

IgLchain genes.

In contrast, ALLs wereinitiallyclassifiedaspre-Bcell in type ifthey

lackedTcell associatedantigens,demonstratedBcellassociated

antigens

(B4,HLA-DR, andattimesB.), possessed rearranged IgHchain genes andattimesrearrangedLchain genes(2, 3). Completeexamination of all such markerswereperformedwhenever

possible.

Limited numbers of leukemic cellsprevented examination of all markers insomecases

and such cellswere classified aspre-B orTbased onavailable data

(Tables

I, II).

TCR geneconfiguration.Highmolecular weight DNA was extracted from leukemic cells or cell lines as well as total white blood cells (WBCs) from normalindividuals. 15ygof genomic DNA was digested to com-pletionwith 5-10U/pgDNAofthe appropriate restriction endonuclease,

sized fractionatedon0.6-1.0% agarose gels, and transferredto nitrocel-lulose paper(2, 45).Such genomic blots were hybridized to 32P-labeled DNAprobes

prepared

by a randompriming method (46). These Southern blots were washedatthe appropriate stringencyandautoradiography wasperformed.

The

Cp

probe was a 700 base pair (bp) Eco RI cDNA fiagment

con-tainingthe murine

Cp

regionand iscapable ofhybridizingtoboth human

Cp

regionsunderroutinestringency(14). In the germline form both Jr

Cp

Iand

JrC1,2

arelocated onasingle 23 kb Bam HI

fiagment

asindicated inFig. I A. Thus, rearrangements of either the

C1,l

or

Cp2

complex should alter the size of the Bam HI

fragment.

Todiscern theidentityof such rearrangements EcoRIandHind IIIdigestswereused.Inthe germ-line form

C#l

occupies an 11-kb EcoRI

fragment

and

C,2

a4-kb frag-ment.The 11-kb fragmentcontains the

J,0l

region and rearranges when the

Cp

lcomplexisused,whereasthe4-kb fragmentlacksJ,2andnever

rearranges.Occasionallyan8-kb Eco RI band is noted eveningermline sourcesoftissue.This representseitheranEco RI resistant site or perhaps

attimesarestriction fragment length polymorphism, andcannotbe assumedtobeasomaticrearrangement. In contrast, Hind IIIdigestion

is capable of detecting rearrangements of the

Cp2

locus.

JrC#2

occupy

an8.0-kb Hind IIIfragmentthat rearranges.

C#l

isfound on a 3.5-kb Hind IIIfragmentthatis deleted whenaV, regionrearrangesinto the

Cp2

locus.

The

C,

probewas a300 bpPstI-BamHIfragmentofahuman

Cy

containingsubclone(30).Itwasroutinely hybridizedtoBam HIdigested

humangenomicDNAand thosecellsdisplaying germline patternswere

alsoexamined withSac I and PvU II

digested

DNA. Initsgermline configurationtheC7 locusdisplaysa15kb Bam HIfragment containing

C41

and a12.5-kbfragmentwith C72 (28). Bam HIdetectsrearrangements ofeitherthe

C71

orC72locus.AsinglesetofV7 genes appearstoexist

5'ofC71 asdiagrammedinFig.3 B.Hence, rearrangements into the

CY2locus result in the deletion of the 15-kb

fragent.

TCR gene expression. In addition RNA was extracted from 14 T cell ALLleukemic samples and cell lines, 4 pre-B cell lines,4mature B celllines,Hut102,HL-60, and K562. Total cellular RNA was prepared

byaguanidinethiocyanatemethod and10

,gg

wasdenaturedin form-amide,electrophoresedonagarose-formaldehyde gels, andtransferred

tonitrocellulose paper(47). Northernblotswerehybridized with the C. andC, probes describedaswellaswitha1.1 kb Eco RIfiagment con-tainingCa from ahumanaTCR cDNA clone(23). In addition all Northern blotswerehybridizedwitha-y-actinprobe to insure that equal

amountsofintact, hybridizableRNAwaspresent in each lane(48). Blots werestrippedclean of probeby washing in0.1%sodium dodecyl sulfate

(SDS)at70'Cfor 20 minand autoradiographywasperformedtoinsure acompletewashoff.

Ig geneconfiguration.HumanIggeneprobes were used to assess the

statusof H chainaswellas Kand A Lchain genes.Ahumangenomic

6.0 kb Bam HI-HindIII

fragment

containingtheentire JH regionwas

usedto assessrearrangements in both BamHI andHindIIIdigested DNA(49). Kgene rearrangementsweredetected inBam HIdigested

genomicDNAwitha2.5-kbgermlineEco RI

C,

containing fiagment

(50). A0.8 kb BglII-Eco RI fragment containing C,, wascapableof detecting anyXrearrangements in EcoRI digestedDNA(50).

Phenotypic characterization.Whenadequate numbers of leukemic cellsexisted,theywereassessed for cellsurfaceantigens.Onemillion viable cells pertest werereacted withapredetermined concentration of

a mousemonoclonalantibody at4VC for 30 min, washedtwice,and thenreacted withafluorescein-labeled goat mouse Ig second anti-body. After three washes the cellswereassessedby flowmicrofluorometry

witha fluorescence-activated cellsorter(51). Cellswerereacted with normalmouse serum and thefluorescein-labeled secondantibodyas a

backgroundcontrol.Afluorescence

profile

ofeach

antibody

wasproduced

(4)

ofthe percentageofcellswithin the leukemicpopulationthatbore the antigen. Alternatively, a few samples were assessed by direct visualization withconventional immunofluorescence microscopy. If > 50%o of the cells bore the antigen, the monoclonalantibodywas scored aspositive (+), whereas<10% was scored asnegative, and values between 10and50% were listed in Tables I andII.

Monoclonal antibodies used here include the 3A-Imonoclonal that recognizes CD-7 (52),T1I (CD-2) (53), T3 (CD-3) (54),T4(CD-4) (54), T8 (CD-8) (54), T6 (CD-l) (54), B4(CD-19) (3), Bl (CD-20) (3), J5 (CALLA, CD-10)(55), and I2(HLA-DR) (56).Inaddition many of the cellswere analyzed with BA-1 (CD-24) (57),BA-2 (CD-9) (58),T12

(CD-6)(54), and for terminaldeoxynucleotidyl transferase(tdt)(data

notshown). CellslackingIg and TCR gene rearrangementswereexamined for myeloid-associated antigens with M02 (59), LeuMl (60), and LeuM3 (61).

Results

13

TCR

gene

configuration

in T

cell

ALL.

The

vast

majority

of

patients cells that would phenotypically beclassified as Tcell

type ALLpossessed rearranged(3TCR genes(23/26) (Table I,

Fig.

1). Most cells rearranged both alleles

(19/23) of their

1-chain genes, whileoneexceptionalcasedisplayedan

unantici-pated three rearrangements

of

1 TCR restriction

fragments (Fig.

1 B).

Cytogenetic examination

of

this

cell revealed numerous

abnormalities

including

a

translocation, t(7: 11) (q35-36; q14)

at or near

the band

containing

the13

TCR, 7q35.

Such an

extra-rearrangement

could

conceivably

be created

by

achromosomal translocation

breakpoint

that fell within the

CB

TCR

region.

Alternatively, this might

represent clonal evolution or a

dupli-cated TCR gene that

subsequently rearranged.

This leukemia

provides

an

opportunity

to

explore

these

possibilities. By utilizing

three

restriction

endonucleases, Bam HI, Eco RI, and

Hind

III, it

is possible

to

discriminate

between rearrangements of

Ce

I ver-sus

the

C,02

regions (Fig.

1

A-D).

All three rearrangements in

patient

4 were

of

the

C,2

complex

(Fig.

1 B). Incontrastboth rearranged

alleles of

CEMwere

Cp

Iand the

CB2

locus was

germ-line (Fig.

1 C). Rearrangement

of

C,0

and

C,62

loci

were not

exclusive, however,

as

patient

2 and several others

revealed

a rearrangement

of

C,61

onone

allele

and

C,02

onthe other

(Fig.

1

D).

Overall,

therewas no

preferential

usage

of

Cp

I versus

C'02.

Moreover, therewasno

correlation between

Cp

1versus

C62

usage and the

cell surface

antigens expressed

orany

postulated

devel-opmental

status

(Table I).

13

TCR

geneexpression in T

cell

ALL. MostTcell ALLs with rearranged13 TCR genes also expressedthis locus (11/13) (Table I and

Fig.

1E). Nearly all cases that expressed13produced the

full length

1.3-kb

transcript (10/11) signifying

the presence

of

a

completed V/D/J

rearrangement. Four cellsexpressed the 1.0-kb RNA

compatible with

the product

of

anintermediate D/ J rearrangement. Three

of

these also expressed a

full

sized

tran-script

while

one T-ALL

possessed

only

the truncated

1.0-kb form.

13TCR gene

expression

was not

completely

lineage restricted

as Raji, a Burkitt lymphoma cell line, produced a truncated 1.0 kb

13

transcript

(not

shown). Four of the Tcells that expressed13

failed

toexpress theaTCRsupportingasequential use of these genes.

a TCR gene expression in T

cell

ALL. The extraordinary

long

stretch

of

genomic Ja regions

makesit currently rather

im-practical

to

conclusively

determine the status ofa TCR rear-rangements in every T cell. For this reason we concentrated uponaTCR locus

expression.

Incontrast tothe13TCR

expres-sion, slightlyoverone-half (8/14)of the T-ALLsexpressedthe aTCR locus (TableI,Fig. 2). Most oftheseTcells (7/8)possessed

the full length 1.6 kb transcript. Most T-ALLs with a TCR expressionwereofthe most mature type displaying surface T3. Mostof these (7/8) also expressed a 1.3-kb1 mRNAand pre-sumably possessedafunctional T3-Ti complex. One important exception expressed yandaunique 2.0 kbaTCRgenebutnot 13 (patient 7). In addition one case expressed a, 13, and y TCR genes(patient 4). No individuals expressed a alone, compatible with a developmental sequencewherea-chain production isa

later eventin T cell ontogeny. a TCR gene expression is not lineage restricted as RPMI 8432 a B cell lymphoblastoid line (Fig. 2), SU-DHL-6 a mature B cell and RS a pre-B cell (not shown) were all noted to make a truncated 1.3-kb form of the

atranscript.

yTCR gene rearrangement in TcellALL.MostTcellALLs

(23/26)

have rearranged their -y TCR locus

(Table

I,

Fig.

3 A, B). Moreover the vast majority of T-ALLs that utilized y,

rear-ranged both alleles (17/23). Examples

(4/23)

occurred in which

either one allele had been deleted or twoseparate rearrangements fortuitously createdthe same sized Bam HIfragment. Inspection of y TCR rearrangementin T-ALLs revealed thatapparently all hadrearranged into the Cz2 complex. In general T-ALLs

show noremaining

C.y

1 containing 15.0 kb BamHIgermline fragment and no remaining

C72

containing 12.5 kb Bam HI

germline fragment (Fig. 3 A). This findingresults from

rear-rangementsinto the

C.Y2

locusonboth alleles(Fig. 3 B). This

rearrangement pattern in T cells is different than the y TCR rearrangementsseenin pre-B cells

(Fig.

3

D)

aswill be

presented

below. Unusual patterns included

patient

16 with three

rear-ranged

Cy

fragments. Unfortunately,

no

cytogenetic

data is

available

on

patient

16. Overall 22 of 26 T-ALLs

rearranged

boththeiry aswellas 1 TCRloci and the

important

exceptions will bedetailed later.

y TCR gene expression in TcellALL.

Despite

the

frequent

rearrangement ofy TCR genesinT-ALLs this genewasonly

occasionally

transcribed atdetectable levels

by

Northern blot

analysis

of total RNA.

Only

3 of 14 T ALLs examined

(patients

4and7and the HSB cell

line)

revealed anyy

transcripts (Fig.

3

C,

Table

I).

Allthree

of

theseT

cells possessed

y TCR

rear-rangements.

,y

Without

13,13

without

y TCR rearrangement. Several cells

possessed

rearrangement patterns thatargue

against

any

strictly

fixed

order to13andyTCR gene rearrangement. Two rearranged

-y

TCR genes in the presence of

germline

1 TCR genes were

evident in patient

7

(Fig.

4 A,Table I).

Curiously, this

cell

dem-onstrates a rare y TCR pattern fora Tcell in thata

germline

12.5 kb Cz2 band

is

retained arguing that one

of

the

rearrange-ments

involves

the

ClyI

locus. It also expresses

-y

TCR RNA

(Fig. 3 C). This

cell also

displayed

a

germline

pattern

for

Ig heavy

chain

andKandX

light chain loci. However, this

T-ALL

did

notappeartorepresentan

early

cell in

differentiation

asit

expressed

a2.0-kbaTCRand

displayed

surface

T3

(Fig. 2,

Table

I). This

illustrates that13 rearrangement is not

required for

a TCR gene

activation.

Moreover, the presence of cell surfaceT3 in the absenceof13 TCR gene rearrangementor

expression

in-dicates the presence ofaT3 receptor

complex

withoutaclassic a,13heterodimer.

The

opposite

TCR geneconfiguration was demonstrated by

patient

3which displayeda

1-chain

rearrangement butgermline

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- t_4w -23Kb _

--8Kb

-.ag _ -6.5Kb

4__-4Kb

-!i w ~~~~~~~~~~~-35Kb

E

13kb-

1.Dkb-Figure1.(A) Schematic presentationof the human,BTCR locusand

theutilizedBamHI,Eco RI, and HindIll restrictionsites. Variable

(V), diversity(D),joining(J),andconstant(C)regionsareshown. (B)

, TCR configuration in patient4 that shows threereangments

(ar-rows) of the

C,92

complex when comparedtothe germline configura-tion(dash marks) revealed by thecontrol. The additional8 kb Eco RI

fragmentisseeninthecontrol andmayrepresentanEco RIresistant

t' 4t 't <

i z-- -13kb

5 w ~~~~~-1.0kb

site.(C) , TCR pattern ofCEMdemonstratestworearrangements of

theC~l complex. (D)

P

TCRpatternofpatient 2 illustratesone rear-ranged

Cpl

andonerearranged

C,2

allele.(E) Northern blotanalysis of total RNA from T ALLpatientsatdiagnosis(Dx)orrelapse (Rel)

and three T-ALL lines. Fulllength (VDJ)1.3kb and intermediate (DJ)1.0 kbtransciptsarenoted.

B

Bam Hi

1Kb

4_

-23Kb

EcoRI

I.

0

Hind111

Cie

c?"

L..A.

lfi

~,I

..* .. E

-4Kb

-8Kb -6.5Kb

D

BamHI

8%

-8Kb -6.5Kb

-3.5Kb

EcoRI

eV

41i6

4v

do

Hindill

8%

to'

Ne

Ir

e. e0 e

.,*-- 4~", !

I

--.> 4w

(7)

eee e e e e ~~~~~A,

4t: 4- 4t 4t' t4 t C' Z !~j

Probe

CTr

1.6-Probey Actin

--.-s

-U

S.

Figure 2. NorthernanalysisofT-ALL atdiagnosis(Dx) plus three

T-ALLcelllines (CEM, 8402, HSB),one mature Tcellline (Hut-102),

and an EBVtransformedBcellline(8432).Fulllength 1.6-kbaTCR

transcriptsarenotedbydashmarks,and the 8432Bcellline revealsa

truncated 1.3 kb RNAyactinhybridizationis shown below.

kb Bam HI

CY

containing fragments

were

of

approximately equal

intensity

in

patient 3 suggesting

that

there

was no

deletion

of

the y locus

either.

No rearrangement was seen

with

Sac I or PvU II

digests

either (data

not

shown).

No RNAwas

available

for

evaluation

of

TCR gene

transcription,

butthe

T3 molecule

wasabsent

indicating the

lack

of

a

complete

T3-Ti

complex.

This cell

suggests

that

(3 can rearrange

without

preexisting

y rearrangements.

Several

other

leukemias

demonstrated

atypical

combinations

of

TCR gene

expression

and cell

surface

antigen phenotype

worth

noting (Table I).

T-ALL

patient

17

clearly expressed

1.3

kb (3

TCR

RNAand

displayed

T3,

yet

lacked detectable

a

TCR

RNA.

While

we cannot

exclude

the presence

of

small amounts

of

a RNA

it

is conceivable

that

this

Tcell hasa

T3

receptor

complex

without

an

a-chain

contribution.

In contrast,T-ALL

patient

11

expressed both full

length

aand (3 TCR RNA yet

demonstrated

the

T3

molecule

on

only

7%

of cells.

Thus,

this

T

cell

may possess

either

a

defective

a or (3 TCR

chain

or

alternatively

a

deficit

inone

of

the

various

components

of the T3 molecule.

ALLs

germlinefor

both

TCR

and

Ig genes. Two ALLs

lacked

rearrangement

of either Ig

orTCR genes.

Patient

5had

germline

Ig

heavy,

K, X genes as well as

germline

y and

(3TCR

when

assessed

with three

enzymes Bam

HI,

Eco

RI,

and Hind

III

(Fig.

5A, Table

I).

This cell

expressed

cell

surface

3A-1

(91%),

but lacked themore

definitive antigens of

Tcell

commitment

such

as

T1I

orT3. Asmall percentage of the

leukemia

cells

actually

expressed

myeloid

associated markers Mo2

(6%),

LeuM1

(27%),

and LeuM3

(7%)

butfailedtodemonstrate

myeloperoxidase.

Patient

18 also demonstrated

entirely germline configurations

of Ig

and TCR genes and

expressed

cell

surface

3A-1 as

well

as

T1I

(Fig.

5 B, Table II). The presence of y and (3 TCR gene rearrangement in several T-ALLs with 3A-1, butno T

1I

suggests

that the

acquisition

of T.,

may

frequently

follow

yand

(l

TCR rearrangement

(Table

I).

However,

patient

18indicates that

this

developmental

sequence is not fixed as

T.,

is present in the absence

of

gene rearrangement. Patients 18 and5represent very

immature

leukemic

cells thatare

uncommitted

atthe

Ig

and TCR gene level.

Lineage

spillover

of

Ig

and

TCR gene rearrangements. We

examined the

status

of

TCR genes

in

what had been

classified

as

pre-B

cell

ALLand

conversely

the

Ig

genes in

leukemias

cat-egorized

asT

cell

ALL.

This

comparison

revealeda

higher

in-cidence

of TCR

rearrangement

(especially

-y)

in

pre-B

cell ALL than

Ig

rearrangements

within

Tcell ALL

(Tables

1-111).

ALLs were

classified

as

pre-B

cell

in

type

if

they expressed

surface

B4,

HLA-DR,

as

well

as

rearranged Ig

heavy

chain

genes and lacked

T

cell

associated

antigens (Table II).

A

portion

of these

pre-B

cells

also

displayed

BI and had

progressed

toKorX

light

chain

rearrangement.

This

pre-B

cell ALL group had

rearranged

y

TCR genes

in

eight

of

17 cases

(Table II, III). Moreover,

the presence

of

light

chain

generearrangement

did

not

preclude

Y

TCR

rearrangementas

pre-B

ALL

patients

1,

2, 4,

and 17

rear-ranged

both

sets

of

genes.

However,

the overall pattern

of

'y

TCR

rearrangement in the

pre-B

ALLswas

different

than that

observed in the

Tcell ALLs

(Fig.

3

D).

Pre-B ALLsat

times

rearranged only

a

single

y

TCR

allele

(5/8).

Incontrast to

the

T-ALL pattern

(Fig.

3

A)

the

12.5-kb

Bam HI

fragment

con-taining

germline Cz2

is retained in the

pre-B

ALLs

with

y

rear-rangements

(Fig.

3

D).

Eventhe

pre-B

cells that

rearranged

both

,y

TCR alleles retained

one or

both

copies

of

the

germline

12.5

kb

C.2.

Thus,

the

pre-B

ALLs rearrange the C 1

complex

fre-quently

incontrast tothe T ALLs that

rarely

use

it

and

pref-erentially

rearrange

into

Cz2. Moreover, pre-B

cells

with

yTCR rearrangement

frequently

retained

germline

#

TCR genes

(6/8)

as

contrasted with

the

generalized

coordinate rearrangement of

both (3

and

y

in

T-ALLs.In

addition,

,B TCR

rearrangements

werenoted

in

3

of

17

pre-B

ALLs

examined

including

one

ex-ample

in which

, was

rearranged

but y

TCR

geneswere

germline

(Tables

II,

III).

We

wished

to

determine

if

the

particularly

high

frequency

of

yTCR

rearrangement

in

pre-B

cell ALLwas

peculiar

to

this

leukemic subset of

cellsor

reflected

ahigh

incidence

of

y

TCR

rearrangements within the entire

B

cell

lineage.

To

resolve this

we

examined

12

neoplasms

of

matureBcell

phenotype

aswell as 14

clonal

Epstein

Barr

Virus

(EBV)

transformed

mature B cell

lines.

None

of

these 26matureB

cells

possessed rearranged

y

TCR

genes

(Table III).

4

of

the 26matureB

cells

rearranged

one

allele of their

(

TCR

genes

(Table

III). Thus,

the

incidence

of

(

TCR rearrangement

in

pre-B

cells

(3/17)

versusmatureB

cells

(4/26)

wasnot

grossly

different.

However,

the

frequency

of

y

TCR rearrangements in

pre-B

cells

(8/17)

wasmuch

different

than in

matureB

cells

(0/26). Therefore,

there does

notappear

tobea

generalized

high

rate

of

yTCR

rearrangement

in theB

cell

lineage

as

rearrangements

werenot

found in the

matureB celltumors.This

indicates

that the

pre-B

cellALLsare adistinct

leukemic

subset and donot

represent

thesame

progenitor

cells that generate themature

neoplasms.

Transcription

of

thea TCRlocus

is

not

lineage

restricted

either. Truncated 1.3-kb amessageswere

found

inmature B

cell

lymphoma

cell line

SU-DHL-6,

EBV Bcell

line

8432,

and

pre-B

cell

line

RS

(Fig. 2,

Table

III).

In

addition,

even a

truncated

1.0-kb

fl transcript

was

present

in the

Raji

Burkitt

Bcell

lym-phoma

cell line.

Consistent with

our

prior

studies, Ig

Hchaingene

rearrange-mentwas

observed

insomeTcellALLsbutatalow incidence

(3/25)

(Tables I,

III).

None

of

theTcells

examined

had

rear-ranged

their

KorX

light

chain

genes

compatible

with this

being

(8)

A

15Kb-

12.5Kb-0~) ,%. ~

)

4t

4

<t

<

C)

15Kb--15Kb

12.5Kb--12.5Kb

...

~~~~~~a

15Kb-15Kb- -15Kb

12.5Kb- -12.5Kb

12.5Kb-C,

*,

C

A-'

0s

-15Kb

k- a | = L

12.5Kb

-15Kb

slFw -r~1.5K

W%

*

O 0)i ~

BamHl

V

1kIb

1 kb

BamHl BamHl

i I

Pi PI VA

m- CyProbe -[

D

15Kb-

1.-oSP

t~°qN q< zo

P -15Kb

". _" -12.5Kb

-15- _ ra

125-4

*:A:

..1

...

ProbeyActin

Figure 3. (A) yTCRgeneconfigurationinT-ALLsatdiagnosis (Dx)

orRelapse(Rel)andintwoT-ALL lines. BamHIdigestsprobed with

aC'y probe placetheC1.l complexona15-kbfragmentand

C,2

on a

12.5-kbfragment. Rearrangements(arrow)of bothallelesare

com-mon.Deletionof both germline

C,

landC72bands indicatesanearly

uniformusageof the C72complex.(B) Schematic presentation ofthe

human yTCRlocus and the BamHIrestrictionsites. (C)Northern

analysisof T-ALLs andTALL cell linesplusHut 102 for

COy

expres-sion.Fulllength1.5 kb'yTCRtranscriptsareindicatedbythedash

marks. 7y-actinhybridizationis shown below.(D)yTCR

configura-tion inBamHIdigestedDNAfrompre-Bcell ALLpatients.In

con-trast totheT-ALLs, thepre-Bcells retainagermlinecopyof

C,2

(12.5 kb)whentheyrearrange.

B

BwnHi

J2 C2

F.3 PA Probe

CTY

(9)

Bam HI EcoRI

11 Kb- sw- 8.0Kb-- VW

6.5Kb-4Kb-

3.5Kb-CTP

23Kb-CTP

Bam

HI

,

:i.

+WTPW

12.5Kb-

04_

' '*

1,

TI,

and usually T3 expressed the a TCR. The T ALLs that expressed theaTCRalso expressed

,3

TCR with one important

HindIII

exception (Table

I).

Most T-ALLs areof sufficientmaturity to

I have rearranged both theiryand(3TCR genes and these events

in

general precede

a

TCR

expression.

Most T-ALLs that

rear-ranged

(3 in our study and in that by Sangster et al. (62) also

expressed it suggesting

that once rearranged, the(3 TCR locus

continues

tobe

expressed

throughout development.Incontrast

only occasional T-ALLs that rearranged the y TCR actually

expressed

it

(3/14)

indicating

thatitmaybe

expressed

only during

anarrow

window

of development.

It

is

possible that this gene

is

vestigial

by

the

stage

of

Tcell

development

thatmost

of

these T-ALLs

reflect. This is consistent with

aproposed murine se-quence

in which

y

expression

precedes a, while( is expressed

throughout all of thymic development (33-36). Curiously, the

CTP

T-ALLsusually haverearranged both y TCR alleles and have

almostalways

utilized

the

C72

complex

(Fig.

3A).

Interestingly,

deletions of

Cy

alleleswerealso observed. Such deletion events have

previously

been noted within IgHchain or K L chain loci and may be

involved

in

allelic

or

isotypic exclusion

(2, 50).

While

most T-ALLs had rearranged bothyand(3TCR genes important

A

BamHI

40

t odo

4-Bam Hl EcoRI HindIII BamHI

*oat F S~s

oIN

*

m.

eCJ e d

CTP

Figure4.(A)Patient7reveals therarepatternofyTCR

rearrange-ment,butgermline TCRs.(B)Patient3 reveals the unusual TCR

configurationinwhich rearranges,but yisretained in thegermline

form.

tinE-11Kb

-15Kb

-4Kb

CTY

CTO

CT3 CTP JH

alater step inBcelldevelopment. Thus,thefrequencyofyTCR

rearrangementspilloverintothepre-BcellALLswasalsomuch greater thanthe frequency ofIgrearrangements within T cell ALLs.

Discussion

Developmental

hierarchy

of

TCR activation andTcellantigen expression. Thedetermination ofa, (3, yTCRgenestatusplus

Iggeneconfigurationcanbecombined withlineageassociated surfaceantigenstoclassify nearlyall ALLsaspre-BorTin type. The extensive characterization of TCRgenesinthisstudy

re-vealed thattheiractivationisnotsolely restrictedtotheTcell lineage,justasIggenerearrangement isnotrestrictedtoBcells.

However,the T cell ALLs did revealarough hierarchyof TCR activationeventsand cellsurfaceantigen expressionwithin the Tlineage (Fig. 6). Onlythemost matureT-ALLs which had

3A-B

BamHI

$ C.

e C

BamHI

$ C.

EcoRI

C.

Hindil eCJ

Ne A -23Kb -# -11Kb

*o -15Kb

w -12.5Kb

SiS -4Kb

Bam HI

4.7

....l.L

-8Kb

A-17Kb

-6.5Kb

.XK..b

-3.5Kb '

i,

CTY CTP CTTP CT3 JH

FigureS.(A)Patient5demonstratesgermline'yand

#

TCRgenesas

wellasIgH-chaingeneswhenexaminedwith theJHprobe. (B)

Simi-larly, patient18isgermlineforyand

#

TCRandIgJHgenes.

552 Felix, Wright, Poplack, Reaman, Cole, Goldman, andKorsmeyer

A

BamHI 4,°,z'

0? t

15Kb -* 12.5Kb-- -* is

CTY

B

Bam

HI

15Kb- J4#$- A

. -8Kb

R.. * -

-6.5Kb

Gl

4

Tr -17Kb

I

*

-3.5Kb

(10)

23Kb-Table IL B-Precursor ALL Patients

TCR Genes Ig Genes Phenotype*

Patient

No. CTY No. CT# No. JH No. Ck No. CA No. 3Al T1 1/SRBC-R T3 B4 DR Calla BI

Bam HI

1 G R 1 R 1 R 1 - + + 11

-2*

R/D 1/1 G R 2 D 2 R 1 - - - + + +

3 R 1 G R 2 G - - + + + +

4 RID 1/1 G R 1 R 1 - - +

5 G G R 1 G - - + + +

6 G G R 2 G - - 49 + +

7 R 2 G R 1 G - - - + + + 22

8 G G R 2 - - - + + + +

9 R 2 R 2 R 1 G - - - + + + 49

10 R 1 G R 1 G - 32 - + + 35

-11 G G R 2 G G - - - + +

-12 G G R 1 18 22 45 + -

-13* G G R 2 R 1 G - - + + +

14 R/D 1/1 G R 2 + + + 46

15 G G R 1 R 1 - - - + + -

-16 G G R/D 1/1 G G 12 30 - + + +

17"§

R 2 R 2 R 2 R/D 1/1 G 32 25 + + +

*R,rearranged, D, deleted, G,germline,No., number of alleles. Data in whicha(-) is<10%of cellsshowing reactivity,a(+) is>50%

reactiv-ity,andspecificvaluesaregivenfor the 10-50% range. IPatient 17's WBCwasonly7,800and but53% blastsatthetime ofthisstudy. The3A-1 reactivity (32%) and T3 reactivity (25%)werecontributed bycontaminatingnormalTcells. *TheBcellassociated monoclonalantibody BA-I

was substitutedfor B4 in these cases.

exceptions

were

discovered.

Examples

of

y

without

,3

TCR rear- that thereis no strict orderfor the initiation ofy and

#3

TCR rangementand also

,3

without My TCR rearrangement were noted rearrangement. However, we can not exclude thepossibility that here

(Fig.

4 A,B).Similar configurations were present in the B unanticipated regionsof the yTCRregion mightexist that can cells

bearing

TCR rearrangement

(Table

II). These cells argue not be detected at present. Theearliest T cellassociated antigen

toappear is the3A-1 molecule although this antigen isnot

com-pletely

restricted

to Tcells

(Fig. 6) (52).

The

T11

or

sheep

red Table III.Lineage spillover blood cell (sRBC) rosette receptor follows and T3 is present

fol-lowing

the

expression

of theaTCR and the

presumed

generation

T-ALL

patients

andcell lines of the

T,

heterodimer.

Rearranged JH: RearrangedCk:

RearrangedC>,:

B-Precursor ALLpatients RearrangedTCR y:

Rearranged 2 alleles TCR y: Rearranged 1 allele TCR y:

Rearranged I allele TCR -y, deleted I allele TCR y:

Rearranged TCR -y, butnotTCR A: Rearranged TCR

fl:

RearrangedTCR

f,

butnotTCR A: Rearranged TCR -y and TCR

Al:

MatureB-cellmalignancies

Rearranged TCR y: Rearranged TCR

fi:

Rearranged Iallele TCR A: ClonalEBVcell lines

Rearranged TCR y: Rearranged TCR

Al:

Rearranged 1 allele TCR

#l:

3/25

0/21

0/23

8/17

3/8

2/8

3/8

6/17

3/17

1/17

2/17

T-Cell ALL

TCRy Rearrangement

TCRPRearrangement

'TCRsiExpression'

3A-1

0/12

1/12 T3

1/1 Figure 6. Proposed coordinate sequence of TCRrearrangement and expression plus cell surface antigen expression in T-ALL. 3A-I

ap-0/14 pears to

precede

TCRgenerearrangement.-yand

fi

TCRsrearrange

0/14

earlyin development, but in no fixed order. Most T ALLs are mature 3/14 enough to have rearranged both

'y

andflTCR andapproximately

(11)

Unique

T

cell subsets.

Theexceptional T-ALL cases presented in Fig. 4 A, B may representunanticipated subpopulations of T cells

immortalized

by atransformation event. Patient 7 expresses a

unique

a and y TCRtranscript and displays T3 yet neither expresses nor even rearrangesthe

#

TCR complex. This suggests

that

alternative heterodimer

molecules

(ay,

or ax, or

yx)

or even

single chains might

be complexed with the T3 molecule

within certain

cells. This leukemia provides an opportunity to

further define

a

potentially

unique receptor complex that has

also

been proposed

in nonleukemic

Tcell subsets (31, 32). Con-versely, patient 3 rearranges

#

but not

y,

suggesting that y rear-rangement

is

not a necessary

first

event

in

TCR activation.

Lineage

spillover of

TCRrearrangement. Only

10%

ofT cell ALLsin this and prior studies rearranged their Ig H chain genes. Even

this

small group

provides occasional evidence

for lineage

duality.

Forexample T-ALL

patient

16 who rearranged his Ig Hchain gene also had the unusual presence

of

HLA-DR (usually seen onpre-B

ALL). Provocatively,

he

failed

toexpress any

of

the TCR

loci despite

rearrangements

(Table I). Perhaps

sharing

events

of

B

and

T

cell

pathways is reflected by this cell's failure

toprogress

further in

either

B or T

cell maturation.

ALLs that we

would have

categorized as

being

pre-B cell in type

showed

asurprising amount

of

y

TCR

gene rearrangement.

While

-

20% of

pre-B ALLs rearranged

their

(3-chain

genes,

roughly

45%

rearranged

the y

TCR

(Table

II,

Fig. 3 D). Several

lines of evidence

argue that these

cells

are

predominately

Bcell

lineage committed

rather

than being mis-classified

early Tcells.

All of these cells bore

B

cell surface antigens that

are

rarely

or never seen in T-ALLs

including

HLA-DR, B4 and

often B1.

They

had

uniformly rearranged their

H

chain

genes

and

some

had

progressed

to L

chain genes. Moreover,

even

the apparently

out

of

contexty

TCR

rearrangement

displayed

a

different

pattern

in these

pre-B

cells than it did in the

T-ALLs. They

TCR

rear-rangements

in pre-B cells

were more

often

on

only

one

allele

and

frequently utilized C

1, whereas the T ALLs

preferentially

rearranged into the

C72

complex. Thus, by

many

criteria this

group

of cells

differs from the

T

cell

set

and

more

closely

resem-bles the

other

pre-B cells.

Strikingly

however, this

high

incidence of

yTCR

rearrange-ment was not

witnessed in

the matureB

cell

neoplasms and cell

lines. This

group

of pre-B

ALLs

thus

donotrepresent

the

pro-genitor cells that give rise

to thesemore mature

cells.

Once

again,

there

was a

dissociation

of

y

and

(3

TCR

rearrangement

in

pre-and

mature B

cells

arguing against

a

srict order of locus

acces-sibility

and

rearrangement

in

TCR

activation.

It

is

important

that the

high

frequency

of

yTCR rearrangement argues that the

pre-B ALLs

representa

rather distinct

population

of

cells.

Several

potential explanations

for this

unanticipated

event exist. The

pre-B ALLs

with rearranged

y

TCR

might

representa

unique

subpopulation

of

cells thataremade

evident

only by

malignant

transformation

and

subsequent

clonal

outgrowth.

It

is

possible

that the

normal

counterparts

of

these

leukemias

are

precluded

from

developing further

into

either

B or Tcell

pathways. They

lack the Ig

production

of

aBcell yet

their

peculiar

yTCR

rear-rangement pattern

might preclude further effective

Tcell

dif-ferentiation.

Such cells

might

be

confused with

respecttotheir

lineage commitment. Alternatively,

it

is conceivable

that the y

TCRrearrangements are a

secondary

event

following

transfor-mation.

Perhaps these ALLs

reflect early

lymphoid progenitors

lacking either complete

Ig or TCR rearrangements and because

of this

a

putative

common

recombinase

would

remain

active.

By

this

proposal

the yTCR locus

might

be

particularly

vulner-able to further recombination. In this context it would not be surprising ifthe specific pattern ofyrearrangement were different in pre-B or uncommitted cells than in a truly committed T cell.

Standards oflineage

commitment. Considerable spillover

of

Ig H chain rearrangement

within

bonafide mature T cells and

TCR

activation within bona fide

mature B cells have been noted inthis and

previous

studies

(2, 14, 63, 64).

Some

of

theseevents

may be even more pronounced earlier in differentiation. This lack

of

total

lineage fidelity of

these genetic events necessitates some

caution

in

solely

using

these markers to determine a lineage

designation.

Ig L

chain

rearrangement appears to be a later event

in

development and to date hasbeen restricted to B cell devel-opment.

However,

inthe more

immature

pre-B

cells

with

only

H-chain

rearrangement thepresence of the B cell associated

an-tigens

B4

and B1

aswell as HLA-DR are

important.

The lack

of

TCRactivation also

favors

a Bcell assignment, but some pre-Bcells

will

rearrange yor (3andeven express a TCR genes.

One

of the

strongest

criteria for

T

cell commitment in

ALLs

is

the

lack

of

Ig H

and

Lchain rearrangement and the lack

of

the

Bcell

surface

antigens.

The

exclusive

presence

of

rearrange-ments

ofTCRs is

also strong

evidence favoring

a T

lineage

com-mitment,

although the pattern

of

a,

(3,

andyTCR rearrangement and

expression

canbe

varied.

The

3A-

1

antigen is

seen on

early

T

cells, but

is

not

fully lineage restricted,

whereasthe

T1I

and T3molecules appear later in development and are more

defin-itive.

Early

lymphoid

progenitors

within

ALL.

Cells

from two

T

ALL

patients lacked both Ig and TCR

rearrangements

(patient

5

and

18).

One

of these

cells

possessed only

the

3A-1 molecule

and

also

displayed

some

myeloid

antigens

on a

subpopulation

of the leukemic

cells. It thus

appeared

to be

rather

lineage

in-determinant

by several criteria. The other leukemia displayed

T1I

and

3A-1

suggesting

aTcell

identity

buthadnot

genetically

committed its TCR loci.

These

cells

provide

an

opportunity

to

dissect

even

earlier

stages

of commitment in

putative

lymphoid

progenitors. The extensive characterization of Ig loci, TCR loci,

and

surface

antigens

has

helped

define the developmental

state

of individual

TandB

cells,

revealed

unanticipated

TCR

rear-rangement events and clonal

expansions

of unique and

poten-tially

rare

lymphocyte subpopulations.

Acknowledgments

Wewishtothank Dr. Mark Davis for the

P

TCRclone, Dr. Tak Mak for theaTCRclone,and Dr. JonSeidmanfor the

'y

TCRclone used inthisstudy.We also wishtorecognizeMrs.MollyBoschfor her expert assistance in the

preparation

of thismanuscript.

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Figure

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