Acute leukemia expressing the gamma gene product of the putative second T cell receptor

Acute leukemia expressing the gamma gene

product of the putative second T cell receptor.

R González-Sarmiento, … , S Ang, J H Kersey

J Clin Invest.

1987;

79(4)

:1281-1284.

https://doi.org/10.1172/JCI112949

.

Early thymus-derived lymphocytes bearing the T gamma gene product in association with

the CD3(T3) complex have recently been described. We report a unique case of human

acute lymphoblastic leukemia with a CD2+, CD3+, CD4-, CD5+, CD7+, CD8-,

WT31-phenotype. These cells were found to have T gamma gene rearrangement and T gamma

transcripts in absence of T alpha or T beta rearrangement or transcripts.

Immunoprecipitation studies with anti-CD3 antibodies showed a 43-kD protein associated

with T3; this 43-kD protein is also precipitated with antiserum raised against synthetic

peptides representing the constant region of the putative T gamma protein.

Research Article

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Acute

Leukemia Expressing the

Gamma

Gene Product

of the Putative

Second

T

Cell Receptor

RogelioGonzalez-Sarmiento,* TuckerW.LeBien,* J. Garrett Bradley,* JamesM.Greenberg,*J. G.

Seidman,*

SiewAng,t and John H.Kersey*

*Departments ofLaboratory Medicine and Pathology and Pediatrics, University ofMinnesota, Minneapolis, Minnesota 55455; tDepartment ofGenetics, Harvard Medical School, Boston, Massachusetts 02115

Abstract

Early thymus-derived lymphocytes bearing the Ty gene product in association with the CD3(T3) complex have recently been

described.

We report a

unique

case

of

human acute lymphoblastic leukemia with a

CD2',

CD3+,,

CD4-,,

CDS+, CD7+,

(]8-, WT31-phenotype.These cells were found to have T- gene rear-rangement and

_Ty

transcripts in absence of Ta or

Tfi

rearrange-mentortranscripts. Immunoprecipitation studies with anti-CD3

antibodies

showed a

43-kD protein associated

with T3; this 43-kD protein is also

precipitated

with antiserum raised against synthetic peptides representing the constant region of the putative

T'y

protein.

Introduction

Antigen recognition

is mediated in

T

lymphocytes

by the T cell receptor,

identified in

some T cells as aheterodimeric

glycopro-tein comprised

of

disulfide-linked

a-and

_i#-

chains

that are en-coded by

nonlinked rearranging

genes(1,

2). This

receptor is

associated

on

the

membrane of human

T

cells

with the T3

mo-lecular

complex. The T3 complex consists

of

two glycosylated

proteins,

T3'y

and

T36,

and anonglycosylated protein

T3E

(3-5).

A

third

gene

with considerable

homologywith

_Tfl,

designated

Ty,

has beenrecently characterized

(6).

Transcripts encoded by

this

gene have

been detected

in

cytolytic

Tlymphocytes and

early

in T cell ontogeny (7,

8).

The

characterization

of the

T-y

product was recently reported

in

T cells from patients with

im-munodeficiency

syndromes (9) and in a normal thymocyte

sub-population

(10). The new

peptide

wasshown to be associated

with

the normal T3 molecular complex raising the possibility

of

a

putative

secondT

cell

receptor (9, 10).

T acute

lymphoblastic leukemias

(T-ALL)'

posses cell surface

markers

that are very

similar

to those present in subsets ofnormal

Address reprint requests to Dr. Kersey, Box 86, University of Minnesota

Hospitals,Minneapolis, MN 55455.

Receivedfor publication 4 November 1986.

1.Abbreviations used in this paper:FACS, fluorescence-activated cell sorter,PMSF, phenylmethyl sulfonyl fluoride; SSC, standard saline citrate; T-ALL,Tcell acutelymphoblastic leukemia; TE, 10 mM Tris, I mM EDTA.

thymocytes (1 1), and this has ledtothe beliefthatleukemic cells

areclonalexpansion ofthymocytes arrestedatdifferent stages of maturation. Herewedescribe the characterization of T-ALL that appearstoexpress theputative

Ty

protein.

Methods

Patient andcells. Leukemic cells were obtained from the peripheral blood andbonemarrowofa13-yr-old male who presented with leukocytosis, hepatosplenomegaly butnomediastinal mass atdiagnosis. Leukemic cellsrepresented 90% of totalperipheralblood mononuclear cells and showedahigh nuclear/cytoplasmicratio and evident nucleolias

mor-phological characteristics. Thepatientwasinduced withmultiagent che-motherapy and has been in remission for 6mo.

Viable cells were phenotyped with monoclonal antibodies and

an-alyzed byindirect immunofluorescence usingacytofluorografOrtho

SpectrumIII,(Ortho Diagnostic System, Raritan, NJ)or afluorescence activated cellsorter(FACS IV,Beckton, Dickinson &Co.,Mountain View, CA).Antigens, andmonoclonalantibodiesused in thisstudy in-cluded:CD1, CD2,CD4,CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD24,CDwl3, CDwl4, and HLA-Dr.Specific antigensof the T cell receptorcomplexweredetected with WT3 1,amonoclonal antibodythat detectsaframeworkepitopeontheTa/, heterodimer(12),and OKT3 (13), and Leu 4 (14), antibodies that

recognize

CD3. A heteroantiserum

(anti-Cy)

raisedagainst synthetic peptides representingasequence of 21 aminoacids(residues136-157)from theconstantregionof the putative

Tgamma protein,wasusedtodeterminethe presence ofTy peptideon

theT-ALLcells(15).

Nonspecific bindingvia Fc receptorswasruledoutbycompetitive bindingstudies.Neoplasticcells were incubated with 20% human AB

serumandanindirect immunofluorescence assaywasthenperformed

toexaminethe loss offluorescentstainingascomparedwithasimilar assay done in the absenceof20% human ABserum.

Radiolabeling andimmunoprecipitationofcell surfaceantigens. Cell surface iodinationwasperformedwithNa'25Iandcatalyzedby

lacto-peroxidaseasdescribed before (16).2X107cellsweredisruptedinlysis

buffercontaining0.5%Nonidet P-40(NP-40;Particle DataLaboratories;

LTD,Elmhurst, IL), 0.1% sodiumdodecyl sulfate(SDS),2 mM phe-nylmethylsulfonylfluoride (PMSF), 1% aprotinin (both from Sigma

Chemical Co., St.Louis, MO),in 0.9% NaCl and 0.1 M TrispH8.1. Afterlysisandultracentrifugationtheresultinglysatesupernatantwas

preabsorbedwithprotein A-Sepharose. Thelysatewasthen useddirectly (forprecipitationstudies withanti-Leu-4),ordenatured(forprecipitation

studies withanti-Cy).Denaturationwasconductedessentiallyasdescribed by Brenneretal.(9).Briefly,thelysatewasadjustedto 1%SDS,2mM

dithiothreitol(DTT),andheatedat68°Cfor 5 min. Itwasthencooled

to roomtemperature,adjustedto20mM iodoacetamide, anddiluted with4volof NP-40lysisbuffercontaining 1.5%Triton X-100. Native lysatewasthenimmunoprecipitatedwithanti Leu-4or controlascitic fluidanddenaturedlysatewasimmunoprecipitatedwithanti-Cyor

nor-mal rabbit serum.Complexeswere then isolatedusingprotein

A-Se-TGamma Leukemia 1281

J.Clin. Invest.

© TheAmerican Societyfor Clinical Investigation, Inc. 0021-9738/87/04/1281/04 $1.00

pharose,and resolved by SDS polyacrylamide gel electrophoresis(PAGE). Autoradiography was conducted as previouslydescribed (16).

DNAprobes.

Tft

gene rearrangement and expression was detected witha770 basepair (bp) complementary (c) DNA constant region probe (17). The

T'y

gene was studied with twodifferent probes. The first, Cg, a600-bp genomicDNAfragment thathybridizes to the constant region ofTy(18) was used to detectTytranscripts. The second, Jg, is a 1-kb genomic DNA

fragment

thatrecognizes J sequences in Ty (19). Ta transcription was detected with Py 1.4, a 1.1 kb cDNA probe that hy-bridizestoTa gene(20).

Detectionofgene rearrangement. High molecular weight DNA was obtained from 2X 107 cells using the nuclei extraction technique as described by Bakhshi et al. (21). After phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamylalcohol (24:1) extraction the DNA was ethanolprecipitated with 2 M ammonium acetate and resuspended in TEbuffer(10 mM Tris,ImM EDTA, pH 7.4). DNA concentration was determined by measurement of the

OD26o/ODD2

ratios. 10

_Atg

of this DNA was digested to completion using restriction enzymes EcoRI, Bam HI, andHind III (Bethesda Research Laboratories, Gaithersburg, MD). Theresultingfiagments were separated by electrophoresis through 0.8% agarose and thentransferredfrom the gel to a nylon membrane (Zetabind, Cuno, Meriden, CT) in 20X standard saline citrate(SSC)using the pro-cedure described by Southern (22). After washing at

650C

in 0.1

XSSC, 0.5% SDS, filters were prehybridized and hybridized as described before (23). The different bands were visualized on autoradiograms after exposurewith twointensifier screens for 48 h at -70'C.

RNAextraction and Northern blot analysis. Total RNA was extracted from leukemic cells andHPB-MLT cell line using the proteinase K method as described by Degen et al. (24). 30

Mg

of total RNA was elec-trophoresed on 1%agarose-formaldehydegels and subsequently trans-ferred to nylon membranes in lOXSSCusing the same procedure as for

DNAtransfer. Prehybridization and hybridization conditions were similar

asfor southern analysis. Transcript sizes were calculated by comparing theRNAmigration withribosomalRNA molecular weight standards.

was ruled out with humanAB serum as describedinMethods. (Datanotshown.)

Southern blotanalysis demonstratedagermline

T(l

pattern inleukemic cells when comparedwithplacentaDNA. This pat-tern, showninFig.2A, after Eco RIdigestion,wasconfirmed with Barn Hiand Hind III digestions (datanotshown). Analysis

of the

Ty

gene withJg,aprobethatrecognizes twoJregions showntworearranged bands of 4.2 and 2.5 kb(Fig. 2 B),

cor-respondingto types IV and Vpreviously described (19,23). We nextstudied the leukemic cells for the presence ofRNA

transcripts.NoTa/a transcriptsweredetected inthe RNAfrom themalignant cells, whereas expressionof both wasclearly de-tected in RNA fromcontrolcells (datanotshown). Northern analysisinFig. 2 C shows the distinct presenceof a 1.6-kB

Ty

transcriptinthe leukemic cells ofthepatient.

Aradioimmunoprecipitation experimentthat wasdesigned

todetect theexpression ofCD3 andassociatedproteinsis shown

inFig. 3.Aprominent precipitatebetween 18 and 30kD,

cor-responding to the CD3 complex is clearly visibleinnormal pe-ripheral T cells (lane 5). Alsoseen inlaneS are bandsrunning

at 40

and

49 kD

corresponding

to the

Ta/(B

heterodimer. Lane4shows theresults obtainedwith anti-Leu4 and the T-ALL cells. The CD3 complexwasprecipitated although,

con-sistent

withthe

FACS

histograms

in

Fig. 1,

theamountof CD3

is less than observed with normalT cells. In contrast tonormal

Tcells, the anti-Leu4 precipitate of T-ALL cells also yieldsa

single 43-kD band. To determine whether the 43-kD protein shownin lane 4 might be similar to the putativeTy protein, immunoprecipitation with

anti-Cy

antiserum wasperformed.

Anintenseprecipitate isseen in lane 1 runningat43kD,with amobility identicaltotheprotein co-precipitatedwith anti-Leu4

inlane 4. Results

The

neoplastic

cells expressed an unusual T cell profile; CD1-,

CD2+, CD3+,

CD4-,CD5+, CD7+, and CD8-. Markers for B

lymphoid,

andmyeloid lineage were negative; CD9-, CD10-, CDwI3-,

CDwl4-,

CD19-, CD20-, CD24-, and HLA-DR-. Fig. 1shows theimmunofluorescent profile of normal peripheral Tcells (A, C)andleukemic cells (B, D) examined for CD3 (A,

B)

and

Ta/a

(C,

D) expression. The leukemic cells were clearly

CD3'

(93%,

meanchannel,

21),

although to a lesser degree than normal Tcells (81%, mean channel, 68). Leukemic cells did not express the

Ta/a heterodimer

asdetected byWT3 1 (4%, mean

channel,

4.Normal T

cells

were 71%

positive,

mean channel,

30).

Nonspecific binding of anti-CD3 antibodies via

Fc receptors

Discussion

TheTygene wasinitiallydescribed in murine Tcells(6)and

subsequently inhuman T cells(18).Thepresence ofTy

tran-A

P c

B

C P

C

c P

11.0KB-

--4.2KB

3.4KB- _

4.0KB- _.Mi _1.6KB

-2.5KB

c _{Figure 1.FACS analysis} ofperipheralbloodT

cellsorpatients leuke-F _{mia cells stained with}

D _{OKT3(CD3)orWT31}

(Ta/a

framework)using indirect

immunofluores-cence.Background

X

staining

has been sub-tractedfrom all histo-grams. Cell number in-creased from bottomtotoponthe

y-axis,

fluorescence

intensity

in-creased fromlefttorightonthe x-axis. Thex-axisisonlinear scale.

8

1.8KB-T Beta T Gamma T Gamma

P'PATEW

C.COTROL

Figure 2.DNAand RNAanalysis of leukemic cells. (A) EcoRI digest probed with

Tfl.

(B) EcoRIdigest probed with T gamma Jg. (C) Total RNA probed withTgammaC. (P, patient; C, control.) Control in lanesAand B,peripheral Tlymphocytes.Control inlane C, HPB-MLT.

T3 WT31 B

68

'p

-43

-31

-21

1

2

3

4

5

6

Figure3.Immunoprecipitation of iodinated surface molecules from leukemicandnormalTcellsusing antibodiestothe T cellteceptor complex. Lanes 1-4,leukemic cells. Lanes5,6,peripheralbloodT

cells.Lane 1,

anti-Cy.

Lane2, normalrabbitserum.Lanes3 and 6, controlasciticfluid. Lanes 4,5,anti-CD3 (Leu4). Molecularmass

markers, inkilodaltons,areshownonthey-axis.The gelwas electro-phoresed underreducingconditions. Immunoprecipitation in lane 4 is weakerthan thatin lane 5 reflecting differences in CD3 expression

be-tweennormalperipheralTcells andleukemic cells and thepresence

ofmorefree iodine in the immunoprecipitate.

scripts

in thymic precursors (but less frequently in mature cells) suggested that the gene product could be functional early in

thymic

development(8). The recent demonstration of the

Ty

geneproduct on the

surface

of cells from patients with

immu-nodeficiency

syndromes(9) and in developing normal thymo-cytes

(10)

isconsistent with this hypothesis.

Here wedescribe the

first

exampleof fresh T-ALL cells with the

CD3+,

CD4-, CD8-, WT3I phenotype that appear to ex-pressthe

Ty

geneproduct ontheir cell surface. These cells ex-pressed the CD3 protein complex as demonstrated by

immu-nofluorescence

and immunoprecipitation studies. However, whenstudied for expression of the

Ta/a

heterodimer,the cells showed no

reactivity with

the

antibody

WT3 1,which recognizes

a

framework

epitope onthe

Ta/(3

heterodimer (12).

Immuno-precipitation

studies under reducing conditions with an

anti-CD3

antibody

showedthe normal

CD3

complex and a unique

43-kD

protein in these cells. When immunoprecipitated with

anti-Cy

antiserum, these cells also demonstrated

the 43-kd

pro-tein. This finding,

along

with

the

existence

of

Ty

transcripts, suggests that thenew

CD3 associated protein

presentin these

neoplastic

cells correspond to the

T'y

gene product.

Biochemical

analysis

of

the putative

Ty

receptor reported

by various

groups have shown

differences

in the protein size. Brenner et al.(9)

studied

cells from patients with

immunode-ficiency

and

described

two

different

CD3

associated

proteins: one,

of

55

kD,

was

immunoprecipitated with anti-Cy antiserum,

the second a 40-kD

protein

was not

precipitated

with

anti-Cy.

Banketal.

(10) examined

thymocytes and

found

aheterodimer

consisting of a 60-kD protein associated with a 44-kD chain recognized bya

Cy

antiserum. Recently, Weissetal.(25) and Moingeonetal. (26) describedtwoadditional55-60 kD and 44

kDprotein associated with CD3. Boththesestudiesshowedthe presence of

_T(l

transcripts, and the relationship of theseto

Ty

isnotclear sincea

Ty

antiserumwere notused in thesestudies. The leukemic cells described hereinexpress aunique 43-kD protein associated withCD3 that is precipitated witha

Ty

anti-serum.However,no

_Tfl

or Tatranscriptsweredetected inthese cells. Moreover, ourstudy indicates that this T-ALL has

_Ty

rearrangementssimilar to that described previously in leukemic and normalcells (19, 23).

Leukemic cells are thoughtto representclonal populations arrested atdifferent stages of hemopoiesis. These cells generally retain fidelityto adifferentiation-linked phenotype although

ex-ceptions have been described (27, 28). To our knowledge, this is the firstreportofaleukemic cell presenting the unusual

CD3',

CD4-, CD8-, WT31-phenotype that expressedaprotein other thanthe Ta/a heterodimer associated with CD3. The precipi-tation ofthis protein byan

anti-Cy

antiserumindicates thatthis

protein is the product of the

Ty

gene. Furtherfunctional,

bio-chemical, and cell culture studies will be done to determineif

thesecells correspond to any functional subset of immunocom-petent Tcells andtofurther define the function ofthis subset.

Acknowledaments

The authors would liketothank Dr.Tak Mak forprovidingTa and

T#

probes and Dr. Michael Brenner forhelpful suggestionsonusing

anti-Cyinimmunoprecipitation.Dr.Gonzilez-Sarmientois therecipientof

aFulbrightFellowshipawardedbytheSpanishCouncil of Education.

Dr.LeBienisascholarofthe Leukemia

Society

of America.

Supported inpartby National Institute of HealthgrantsCA-25097, CA-31685,andtheLeukemia Research Fund.

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