Eur. J. Imm

unol. 1989. 29: 2099-2105 T

cell receptor y/6 repertoire 20YY

Frits Koning,

Marianne K

not, Fred W

assenaar+ and Peter V

an den Elsen+

Departm

ent of Imm

unohematology

and Blood B

ank, University

Hospital, L

eiden

Phenotypical heterogeneity among hum

an T cell

receptor y/&expressing clones derived from

peripheral blood

Hum

an Tcell clones expressing the T

cell receptor (TcR) y/6 w

ere isolated from

peripheral blood lymphocytes of

two unrelated donors. T

he TcR y/6+ clones

derived from one of

these donors were all of

the TiyA

+, 6-T

CS1-, B

B3+

phenotype indicating the exclusive use of the V,9

and Vb3 gene segm

ents. In contrast, the T

cell clones derived from the second donor w

ere either TiyA

+,

6-TC

Sl-, BB

3+;TiyA

-, 6-TC

S1+, BB

3- orTiyA

-, 6-TCS1-, B

B3-.T

he 6-TCS1

determinant w

as expressed on both nondisulfide- and disulfide-linked TcR ylb.

Northern blot and D

NA

sequence analysis indicated that the TiyA

-, 6-TCS1-,

BB

3- clones do use the V

bl gene segment dem

onstrating that the 6-TCS1

monoclonal antibody does not react w

ith all TcR

y/6 using this particular gene segm

ent. In contrast to the 6-TCS1+ T

cell clones, theVbl+

6-TCS1- T

cell clones w

ere found to express Vbl in conjunction w

ith the 563 gene segment suggesting

that this particular Vbl-Jb3 com

bination is not recognized

by the 6-TCS1

monoclonal antibody. In T

cell clones derived from one individual the V

bl gene segm

ent was found to be expressed w

ith either Jb1, Jb2 or Jb3. Heterogeneity

among the 18 clones w

as detected with respect to the expression of the C

D4, C

D5

and CD

8 antigens: one clone was C

D4+

, nine clones were C

D5+ and tw

o clones w

ere CD

8+.Thus, in this panel of clones, heterogeneity exists both w

ith regard to C

D antigen expression and theT

cRy/6 phenotype. A

lso, our results indicate that the 6-TC

S1 monoclonal antibody does not react w

ith all TcR

y/6 using the Vbl

gene segment.

1 Introduction

Tcells expressing a TcR

y/6 have been recently identified in m

an, mouse and the chicken [l-71.

In general TcR

y/6+ cells com

prise a minor population of peripheral Tcells [5,6,

8-11]. In the mouse, how

ever, relatively large numbers of

TcR

y/6+ cells has been found early in thymic ontogeny [6],

in the epidermis [12-141 and in the gut [15, 161. Further-

more, certain V

genes appear to be preferentially used in epithelial tissues [16, 171. H

owever, in the hum

an epider- m

is and gut no large TcR

y/6+ populations are normally

present [ll] and evidence for a limited y and 6 V

gene usage by hum

an PBL

has been found [9, 10, 181.

The physiological relevance of these observations rem

ains obscure since little is know

n about the function of TcR

y/6+ cells, although recently T

cRy/6+ clones have been de-

scribed that

react w

ith (M

HC

-encoded) alloantigens

[19-23). This alloreactivity

might

very well im

ply that T

cRy/6+ cells, like T

cRa/(3+ cells, can react w

ith peptide antigens presented by M

HC

or MH

C-like m

olecules.This is

supported by recent evidence that TcR y/6 cells can specif-

ically recognize (mycobacterial) antigens [24-261. T

he only clear differences betw

een TcR

y/6 and a/f3

cells so far are that w

hereas TcRu/(3+ cells are usually either C

D4+ or

CD

8+, T

cRy/6+ cells are m

ostly CD

4-CD

8- [2, 8-10].

[I 77411

+ Supported by G

rant HY2-YO

from the N

etherlands Organization

for Scientific Research (N

WO

).

Carrespondence: Frits K

oning, Departm

ent of Imm

unohematolo-

gy and Blood B

ank. University H

ospital, PO. B

ox 9600. NL-2300

RC

Leiden, T

he Netherlands

Also, recent studies have indicated that the repertoire of

the TcR y/6 is generated through junctional rather than

combinational diversity [9, 10, 171.

In order

to investigate

the diversity

of cell

surface- expressed TcR

y/6+ and the phenotypic heterogeneity of the cells expressing these receptors,w

e generated a panel of TcR

y/6+ clones from tw

o unrelated individuals. These

clones were

characterized with

regard to CD

antigen expression and phenotype of the T

cRy/6. T

he results show

that TcR y/6-expressing cells are heterogeneous both w

ith respect

to CD

antigen expression and TcR

y/6 pheno- type.

Furthermore, sequence analysis of

a number of

6 chain cD

NA

clones indicates that the 6-TCS1 m

Ab does not react

with all TcR

utilizing the Vbl gene segm

ent.

2 Materials and m

ethods

2.1 Cells and cloning procedures

All T

cell clones described were obtained from

PBL

of two

healthy donors. Following a lym

phopheresis approximately

3 x lo8 PBL

were isolated by Ficoll-Isopaque (Pharm

acia, U

ppsala, Sweden) separation. A

fter washing the cells they

were resuspended in R

PMI 1640 (Flow

Labs., A

yshire, G

B) supplem

ented with

20%

human serum

and 10%

D

MSO

, and stored in liquid nitrogen at lo7 cells/ampoule.

For antigen-specific priming 10’ PB

L w

ere thawed, w

ashed tw

ice and cultured in Iscove’s modified D

ulbecco’s medium

(IM

DM

) supplem

ented with

10%

human

serum

and 10 yg/m

l PPD

(Statens Serum

Institute, C

openhagen, D

enmark) for 5 days follow

ed by continuous culture in the

0 V

CH

Verlagsgesellschaft m

bH, D

-6940 Weinheim

, 1989 0014-2980/8~/1111-209Y

$02.50/0

2100 F. K

oning, M. K

not, F. Wassenaar and €'.V

an den Elsen Eur. J. Im

munol. 1989. 19: 2099-2105

presence of 25 U rIL

2 (Cetus C

orp., Em

meryville, C

A).

Subsequently the cells were harvested, w

ashed twice and

stained for FCM

sorting with the anti-TcR

y/6-1 mA

b (see below

). After separation of theTcR

y/6+ and TcR

y/6- cells, T

cell clones were derived by L

D as follow

s: cells were

plated at 0.3 cell/well in m

icrotiter plates in 0.1 ml IM

DM

containing 10%

human serum

, 0.5%

PH

A (W

ellcome,

Beckenham

, Kent, G

B), 50 U

rIL2/m

l. Each w

ell con- tained lo5 irradiated (2500 rad) allogeneic PB

L and 10J

irradiated (4500 rad) EB

V-transform

ed B cells as feeder

cells. After 7 days another 0.1 m

l IMD

M supplem

ented w

ith 10% hum

an serum and 50 U

rIL2/m

l was added to

each well. A

fter visual identification of growing clones

these were further expanded by restim

ulation with irra-

diated (2500 rad) PBL

in IMD

M containing 10%

human

serum, 0.5%

PH

A and 50 U

rIL2/m

l for 5 days, followed

by continued culture and expansion in IMD

M containing

10% hum

an serum and 50 U

rIL2/m

l until analysis.

2.2 Antibodies

The follow

ing antibodies were used for FC

M analysis:

antLC

D3 (O

KT

3), anti-CD

1, anti-CD

2, anti-CD

4, anti- C

D5, anti-C

D7, anti-C

D8, anti-C

D25, anti-C

D45 (B

ecton D

ickinson, Mountain V

iew, C

A). T

he TcR $6- 1, 6 1, T

iyA

and BB

3 mA

b have been described previously [8, 10, 27, 281 and w

ere the generous gift Drs. J. B

orst, M. B

renner,T.

Hercend and L

. Moretta, respectively. T

he 6-TCS1 m

Ab

was purchased from

T cell Sciences, B

oston, MA

. The

anti-CD

3 6 and anti-TcR y chain antisera are raised against

peptides corresponding to the carboxy terminus of the

human C

D3 and hum

an y chains and have been described previously [29, 301.They w

ere kindly provided by Dr. J. E

. C

oligan.

2.3 FCM

analysis and sorting

For FCM

analysis lo5 cells were stained w

ith the CD

- and TcR

-specific mA

b using previously defined appropriate dilutions for 30 rnin at 4 "C

. After tw

o wash steps, 50 pl of 50

times-diluted

GA

M/lg/FIT

C

(Becton

Dickinson)

was

added for a second 30-min incubation at 4°C

. After one

additional wash step the sam

ples were analyzed using a

FAC

Scan (Becton D

ickinson). Five thousand events were

analyzed in each sample. For sorting 5 X

lo6 lymphocytes

were incubated w

ith 100 yl200 times-diluted anti-TcR

yG-1

ascites for 30 rnin at 4°C. A

fter two w

ash steps, 400 p1 30 tim

es-diluted GA

M/Ig/FIT

C w

as added for a second incubation of 30 rnin at 4 "C

. After one additional w

ash step theT

cRy/6+ cells w

ere separated from theT

cRy/6- cells by

sorting on a FAC

Star at a flow rate of 2000-3000 cells/s.

Sorted cells were collected in 100%

FCS, recovered by

centrifugation and

cloned directly

as described

in Sect. 2.1.

2.4 Radiolabeling, im

munoprecipitation and SD

S-PAG

E

Cell surface iodination w

ith 1251 was carried out as de- scribed

previously [29, 301.

Cells

were

solubilized at

5 X lo8-10 x los cells/m

l in lysis buffer (50 mM

Tris-HC

l, 150 mM

N

aCl,

0.1 mM

PMSF,

10 mM

iodoacetamide,

1 pg/ml leupeptin, 1 yg/m

l chymostatin, 1 pglm

l antipain,

1 pg/ml pepstatin, 0.5%

NP40 or 1%

digitonin, pH8).

Lysis was for 30 m

in at 0°C follow

ed by centrifugation at 13 000 x g for 15 rnin at 4 "C

. Lysates were precleared by the

addition of 100 p1 protein A

-agarose beads (PAA

beads; Pharm

acia) and 100 pl normal rabbit serum

to 1 ml lysate

followed by gentle shaking at 4 "C

for 16 h. After rem

oval of the beads, specific im

munoprecipitations w

ere carried out by m

ixing 10 yl antiserum and 100 p1 lysate for 60 m

in, follow

ed by the addition of 10 yl PA

A beads. A

fter a second incubation of 60 rnin the PA

A beads w

ere washed 4

times w

ith 0.5 ml lysis buffer and analyzed.

Samples w

ere analyzed either on one-dimensional 12%

acrylam

ide SDS-PA

GE

slab gels under reducing conditions or on tw

o-dimensional nonreducing/reducing SD

S-PAG

E

gels as described previously [29-311. After drying the gel

autoradiography was carried out at -70°C

using K

odak X

AR

film and intensifying screens (K

odak, Rochester.

NY

).

2.5 Northern blot analysis

Total RN

A w

as isolated from cells by lysis in guanidinium

hydrochloride [32]. A

pproximately 20 pg of total R

NA

was

size-fractionated in 1.2% agarose gels containing 2.2 M

form

aldehyde and blotted onto Biodyne nylon m

embranes

(Pall Ultrafine Filtration C

orporation, Glen C

ore, NY

). TcR

&specific R

NA

sequences were detected by hybridiza-

tion with cD

NA

probes, 32P-labeled by random prim

ing, as described by the m

anufacturer.The probes w

ere used: Vbl:

EcoR

I/SacI fragment of clone p0-240/38 [33] and Cg

: E

co RI fragm

ent of pGT

CR

2c (l? Van den E

lsen, unpub- lished results).

2.6 Polymerase chain reaction and sequence analysis

Five microgram

s of total RN

A prepared by the guanidin-

ium hydrochloride m

et hod was converted into double-

stranded cDN

A using oligo(dT

) as primer for the first

strand synthesis. Am

plifications of the TcR

6 chain was

performed w

ith 5 U of Taq polym

erase (Cetus) in 100 pi of a

mixture containing 1/20 of the cD

NA

prepared from 5 pg of

total RN

A, 20 pm

ol each of the specific amplim

ers, 0.5 mM

each of dNTP, 4 mM

MgC12, 50 mM

KC

I, 0.6 mg/m

l BSA

and 10 mM

Tris-H

CI, pH

8.4. The am

plimers used w

ere: V

hl: S'-T

GC

TC

TA

GA

GC

TA

CA

TG

CC

AC

AT

GC

T-3';

Vb3:

5'-CG

CT

CT

AG

AG

GC

AG

AG

CT

GC

CC

CT

-3'; C

b: 5'-CT

TA

TA

TC

CT

TG

GG

GT

AG

AA

-3'.

The TcR

G cD

NA

am

plification w

as performed

for 30 cycles. D

enaturation was at 97 "C

for 30 min. T

he amplim

- ers w

ere annealed to the cDN

A for 30 s at 55°C

and the elongation step w

as at 74°C for 30 s. T

he amplification

product was extracted tw

ice with an equal volum

e of a 1 : 1 m

ixture of phenol/chloroform/isoamylalcohol followed by

extraction with an equal volum

e of chloroformlisoam

ylal- coho1 (tw

ice). Following ethanol precipitation the am

pli- fied product w

as digested with X

ba I and Eco R

I.The D

NA

w

as separated on

a 0.8%

low-m

elting agarose gel

in T

AE

-buffer and the approximate bands w

ere cut out. A

fraction of the material w

as ligated into M13m

p18/19 and used to transform

E coli JM

101. Plaques containing inserts w

ere isolated and used for DN

A sequence analysis accord-

ing to Sanger [34].

Eur. J. Im

rnunol. 1989. 19: 2099-2105 T

ccll rcceptor ylb repertoire 2101

1 H6

3 Results

3.1 Isolation of TcR

y/6+ T cell clones and surface

phenotype

In an attempt to generate antigen-specificT

cR y/6+ cells w

e prim

ed lo7 PBM

C from

two healthy donors, know

n to be responders to PPD

, with PPD

for 5 days followed by an

additional 3-day culture after addition of 25 U rIL

2/ml.

Subsequently the cells were labeled w

ith the anti-TcR

y6-1 m

Ab and G

AM

/Ig/FITC

and TcR

y/6+ cells were sorted out

on a FAC

Star. The T

cRy/6+ cells w

ere cloned directly by L

D. In tw

o experiments a total of 18 clones w

ere isolated that w

ere expanded for characterization. A

lthough the clones w

ere raised after an initial priming step w

ith PPD, w

e have so far not been able to dem

onstrate specificity of these clones tow

ards PPD (see also Sect. 4).

Next, w

e surface phenotyped the clones by FAC

S analysis after labeling w

ith CD

cluster and TcR

y/6-specific mA

b. A

s an example the FA

CS profiles for tw

o of the clones investigated are show

n in Fig. 1 whereas the results on all

clones are given inTable 1. Strong staining of all clones w

as observed w

ith the anti-CD

2, anti-CD

3 and the anti-6-1 m

Ab. T

he anti-TcR

y/6-1 antibody also reacted with all

clones although with certain clones only a w

eak staining pattern w

as observed (Fig. 1, compare clone JH

la and JH

6). Most clones stained very w

eakly with antL

CD

8 w

hereas two clones (R

dV4, R

dV5) w

ere brightly CD

8+.

One clone (R

dVw

l) stained strongly with anti-C

D4 w

here- as tw

o clones (JH6a, JH

8) were w

eakly CD

4+ (Fig. 1, Table 1). C

D5 w

as found to be absent on 6 of the 16 clones tested (Fig. 1,T

able 1). Also, the early T

cell marker C

D7

was absent on one of the clones.T

hree antibodies have been described that are thought to react w

ith TcR

y and 6 V

regions: TiyA

most likely reacts w

ith the V,9 V

region [35], 6-T

CS1 w

ith the V6 V

region [36] and BB

3 with the V

63 V

region [37], (nomenclature according to [38]). A

nalysis of the clones w

ith these antibodies demonstrates that the

clones can be divided into three phenotypes:

4 Y Y 8 fi m

3 4

z

JH la

LOG

. FLUO

RESC

ENC

E * Figure 1. FC

M profiles of clones JH

la and JH6 labeled w

ith the follow

ing antibodies: anti-CD

3, anti-TcRy16-1. anti-TcR

6-1 .TiyA

, 6-T

CSI, B

B3. anti-C

D4, anti-C

D8, anti-C

D5 and anti-C

D7.

1. TiyA

+, 6-T

CS1-, B

B3+ (12 out of 18)

2. TiyA

-, 6-TC

S1+, BB

3- (4 out of 18) 3. T

iyA-,

6-TC

S1-, B

B3-

(2 out

of 18)

(Fig. 1, T

able 1).

Table 1. Cell surface phenotype of T

cRy/S+ clones isolated from

PBL

Clone

C

CD

3 y/&

l 8-1

TiyA

6-TC

SI B

B3

CD

2 C

D4

CD

8 C

D5

CD

7

JH 1 la 2 4 5

10 11 12 6 13 2a 6a 8

RdV

3 4 5 wl W

4

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

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

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

+ + + + + + + -

-

-

-

-

-

+ + + + +

+ + + + + + + -

-

-

- -

- + + + + +

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

+ + N

D

ND

?

+

+ +

+ +

+ +

ND

N

D

- + +

+ + f

+ +

+ + - +

f

+ +

+ k

- -

-

-

-

-

-

2102

The five clones isolated from

donor RdV

were all of the

TiyA

f6-TC

S1-BB

3+ phenotype (Table 1). Direct analysis

of the TcR y/6+ cells in the PB

L of this donor indicated the

lack of expression of the 6-TCS1 determ

inant whereasT

iyA

and BB

3 stained similar num

ber of

cells (not shown)

suggesting that the large majority of T

cRy/b+ cells in thc

peripheral blood of this donor is of the TiyA

+, 6-TCS1-,

BB

3+ phenotype. In contrast, all three TcR y/6 phenotypes

were detected am

ong the 13 clones isolated from PB

L of

donor JH

. This correlates w

ell w

ith the finding

that approxim

ately 40% of the TcR

y/6+ cells in the peripheral blood of this donor stained w

ith the 6-TCS1 m

Ab, approx-

imately 40%

with either theT

iyA or B

B3 m

Ab w

hereas the rem

ainder TcR

y/6+ cells lacked reactivity with all three

antibodies (not shown). In conclusion, w

ithin this panel of T

cRy/6+ clones derived from

the PBL

of two unrelated

healthy donors heterogeneity exists both in the expression of C

D antigens and usage of T

cRy/6 V

regions.

F. Koning, M

. Knot, F. W

assenaar and P.Van den Elsen

Eur. J. Im

munol. 1989. 29: 2099-2105

3.2 Biochem

ical analysis of TcR y/6

In order to more critically determ

ine the types of TcR

y/6 expressed

by the T

cell clones

we

analyzed the

cell surface-expressed TcR

by SDS-PA

GE

after imm

unopreci- pitation. A

selected number of T

cell clones, representative for the three T

cRy/6 phenotypes identifed by the FA

CS

analysis, were cell surface iodinated, lysed in digitonin lysis

buffer and after a preclearance step, specific imm

unopre- cipitations w

ere carried out

using mA

b O

KT

3. These

imm

unoprecipitations were analyzed on SD

S-PAG

E under

non-reduced reduced

130-

75-

50

-

39-

27-

17-

12

3 4

12

34

Figure 2. SD

S-PAG

E analysis of anti-C

D3 im

munoprecipitates

obtained from digitonin lysates of

cell surface-iodinated T cell clones. T

he samples w

ere analyzed under either nonreducing or under reducing conditions as indicated.T

cell clones analyzed were:

JHla (lane l), JH

12 (lane 2), JH13 (lane 3) and JH

8 (lane 4).The

samples in lane 4 w

ere run on a separate gel. The low

molecular

mass m

olecules (17-28 kDa) represent the C

D3 m

olecules whereas

the other bands correspond to the TcRy16 chains. T

he position of the m

olecular mass m

arkers (kDa x 1W

3) is indicated on the left.

75 -

50

- 39-

27-

17-

75-

50

-

39-

27-

17-

Figue 3. Two-dim

ensional (first dimension nonreduced. from

left to right; second dim

ension reduced, from top to bottom

) SDS-

PAG

E analysis of anti-C

D3 im

munoprecipitates obtained from

digitonin lysates of cell surface-iodinated T

cell clones. Clones

expressing non-disulfidc-linked y/6 chains are shown in panel A

(JH

8) and panel B (JH

13). Clones expressing disulfide y/S chains

are shown in panel C

(JHla) and in panel D

(JH6). T

he low

molecular m

ass molecules (17-28 kD

a) on the diagonal correspond to the C

D3 m

olecules. The relative position of m

olecular markers

(kDa X

1W3) is identified on the left.

both nonreducing and reducing conditions. The results

(Fig. 2) show that clone JH

la expresses a disulfide-linked TcR

yI6.Very sim

ilar results were obtained w

ith clone JHll

(not shown). In contrast, clones JH

12, JH13 and JH

8 express non-disulfide-linked TcR

y/6 (Fig. 2). Whereas in

clones JH12 and JH

13 only the y chain is clearly visible (45 and 55 kD

a, respectively, see also below), JH

S expresses a 55-kD

a y chain in association with a 6 chain of approxi-

mately 40-42

kDa (see also below

). This 6 chain m

ay contain an intrachain disulfide bridge since it runs slightly slow

er under reducing conditions than under nonreducing conditions (Fig. 2). This observation w

as confirmed by the

analysis on a two-dim

ensional nonreducing/reducing SDS-

PAG

E gel resulting in a 6 chain that is visible just above the

diagonal (Fig. 3a).

Also show

n in Fig. 3 is the analysis of the disulfide-linked T

cRy/6 expressed by the G

-TCSl+ clone JH

6. Disulfide-

linked TcR y/6 expressed by peripheral blood T

cells can therefore be either T

iyA+

, 6-TCS1-, B

B3+ (Fig. 2, lane 1

and Fig. 3c) or TiyA

-, 6-TC

S1+, BB

3- (Fig. 3d). As has

been shown previously, the 6-TC

S1 mA

b can also stain T

cRy/6 that are non-disulfide-linked (Fig. 2, lanes 2 and 3

and Fig. 3b). Thus, biochem

ical analysis of the TcR

y/6 expressed

by this

panel of clones indicates additional

heterogeneity. For identification of the y and 6 chains,

OK

T3 precipitates

of digitonin

lysates of cell surface-

iodinated clones

were

reduced and

alkylated to fully

dissociate all TcR and C

D3 m

Ab. T

he results (not shown)

demonstrate that in the T

iyA+

, 6-TCS1-, B

B3+ receptor

types, the lower chain represents the y chain and the upper

chain the 6 chain. In the case of non-disulfide-linked

TiyA

-, 6-TC

S1+, BB

3- receptors it was found that either

Eur. J. Im

munol. 1989. 19: 2099-2105

Tccll receptor y/S repertoire

2103

the 45-kDa (JH

12) or 55-kDa bands (JH

2a.JH6c, JH

8, JH

13) represents the y chain whereas the low

er molecular

mass proteins (40-42 kD

a) are the 6 chain.

3.3 Northern blot and sequence analysis

Two clones (JH

6a and JH8) did not react w

ith the $6 V

region-specific m

Ab (T

iyA, 6-T

CS1, B

B3) (T

able 1). How

- ever, N

orthern blot analysis revealed that both these clones and the 6-T

CS1+ clones JH

2a and JH12 expressed m

RN

A

containing the Vsl region (Fig. 4, lanes 2-5), w

hereas the control clone (R

dVw

4; BB

3+) lacked expression of theVhl

region at the mR

NA

level (Fig. 4, lane 1). In order to determ

ine the reason for the lack of reactivity of

the 6-TC

S1 mA

b towards the V

al-expressing clones JH6a and

JH8, cD

NA

clones corresponding to the 6 chains expressed by JH

2a, JH6a, JH

8, JH12 and R

dVw

4 were isolated and

sequenced (Fig. 5). In agreement w

ith the results from the

Northern blot analysis, all four JH

-derived cDN

A clones

analyzed contained the Val region w

hereas the RdV

w4-

derived cDN

A clone contained theV

a3 region. In contrast to the 6-T

CS1+ clones JH

2a and JH12, that expressV

b1 in com

bination w

ith the Jb2

or Jal gene

segments,

the 6-T

CS1- clones JH

6a and JH8 expressV

bl in combination

with the Jh3 gene segm

ent (see Sect. 4).

(A)

4 Discussion

2.2 - 1.5-

(B) 2.2 - 1.5-

cs

Va

l

Here w

e report on the phenotype and TcR

repertoire expressed by a panel of T

cRy/6+ clones derived from

the peripheral blood of tw

o unrelated individuals. The clones

described were isolated after a prim

ary stimulation of PB

L

with PPD

. The T

cRa@

+ and TcR

y/6+ cells were subse-

quently separated and cloned by LD

. For none of the

resultingTcR

y/6+ clones we have been able to dem

onstrate specificity tow

ards PPD.T

his is in contrast to simultaneous-

ly derived TcR a

/p+

clones that do specifically respond to PPD

presented by

autologous AP

C (not show

n). We

therefore feel that it is likely that aspecific proliferation of T

cRy/6+ cells w

as induced during the primary culture due

to growth factors produced by the responding T

cRa/(J+

cells (so-called bystander effect).

This is supported by

the observation that the TcR

y/6 phenotypes observed on the clones correspond to the frequencies of

those phenotypes in unmanipulated PB

L

from the donors used for the generation of the clones (not

shown).

12

34

5

Figure 4. Analysis of C

b and Vbl m

RN

A expression by N

orthern blot hybridization. B

lots containing mR

NA

isolated from T

cell clones

RdV

w4

(lane I). JH

12 (lane 2),

JH8

(lane 3), JH

6a (lane 4), and JH

2a (lane 5), were hybridized w

ith Cb

(A) and V

bl (B

) probes.The size of the m

RN

A specics is indicated on the left

(kb).

Tcells expressingT

cRy/6 are usually C

D4-C

D8-

L2, 8-101 although UP to 25%

ofTcR

Y/6+ cells can be C

D8+

[8, 391. In the panel O

f clones investigated in this study two clones

were found to express high levels of the C

D8 antigen, and

V61 N?

D61 N?

D62 N?

D63 N?

GAAATAGT CCTTCCTAC

ACTGGGGGATACG

JH2a TTGGG ...... CCAGTAG.. ..............................

TCC......CGCGAACCA ... ACTGGGGGATACG..CTGGG..........CC

561

JH6a TTGGGGA....AGCCGGC....

............................. CC.. ....... GGCAT....ACTGGGG.....

.. TTCCCTTG ..... CCTGG 563 JH8

TTGGGGA....AGCCGGC. ................................ CC.........GGCAT

.... ACTGGGG.......TTCCCTTG ..... CCTGG 563 JH12

TTGGGG ............................................. CCT .......... C.. ..... CTGGGGGATACG..TCCCTTCGC...CTTTG

562

V63

RdVw4 TGACAC.....TCTGGGA ................................

TCCT. ........ TTT ................................ ACACC 561

Figure 5. Sequence analysis of cDN

A clones corresponding to the S chains expressed by the T

cell clones JH2a. JH

6a, JH8, JH

12 and R

dVw

4 were sequenced. Show

n is the sequence for the junction betweenV

h and Jb only. All clones analyzed w

ere full-length clones within

frame reading fram

es.

2104

one clone high levels of the CD

4 antigen (Table 1). Also, on

a number of clones low

levels of C

D4 andlor C

D8 w

ere detected (Fig. 1, Table l), as also been reported for other T

cRy/6+

clones [22].

All

remaining

clones w

ere C

D4-C

D8-.

It therefore appears that a minor subset of

TcR

y/6+ cells can express high levels of either the CD

4 or C

D8 antigen. In analogy w

ith theTcR

y/6+ cells it might be

expected that those CD

4 and CD

8 molecules on T

cRy/6+

cells can play a role in the strengthening of the interaction betw

een a TcR

y/6+ effector cell and an (MH

C-positive)

target cell. Proper analysis of this awaits the generation of

such clones with a know

n (al1o)specificity.

F. Koning, M

. Knot, F. W

assenaar and P. Van den Elsen

The C

D5 antigen, that is norm

ally expressed on a large m

ajority of peripheral blood Tcells [40,41], was absent on 6

out of the 16 TcR y/6+ clones tested. A

ntibodies to CD

5 can augm

ent Tcell responses [42, 431 and therefore it is thought that the C

D5 antigen could play a role in T

cell responses. T

he absence of the CD

5 antigen on certain clones may

therefore be of consequence for the reactivity of such

clones. How

ever,we also observed that TcR

a@+ clones can

lack expression of the CD

5 antigen (not shown) indicating

that this is not characteristic of a subset of TcR

y/6+ cells only.

FCM

analysis of the clones using theTiyA

, 6-TCS1 and B

B3

antibodies, thought to be specific for theVy9,V

hl and V63

gene products, respectively ([35-371, nomenclature accord-

ing to [38]) indicated that whereas in one donor theT

iyA+

. 6-TC

S1-, BB

3+ phenotype was dom

inant (no other phe- notype detected), evidence for three phenotypes am

ong the

clones derived

from

the other

donor was

found (Table 1). T

his confirms,

at the

clonal level, previous studies [9, 37, 441

indicating that w

hereas in certain

individuals the disulfide-linked TiyA

+G-T

CSl-B

B3+ re-

ceptor type predominate, in other individuals the recipro-

cal phenotype: TiyA-6-TC

S1 +BB

3- (non-disulfide-linked) is easily detectable. T

he present study also indicates the existence of a third phenotype: T

iyA-6-T

CSlPB

B3-.

Biochem

ical analysis showed that the 6-T

CSl+ 6 chain

could be found in combination w

ith three different chains; C

,l (type 1, Fig. 3), the “short form

” of C,2 (type 2a,

Fig. 2), and the “long form”0f C

,2 (type 2b, Figs. 2 and 3). Since these latter tw

o forms of C,2 are allelic products [45],

this indicates that this donor is heterozygous for the C,2 locus.

The results w

ith the clone JH6, that expresses a disulfide-

linked 6-TCS1+ T

cRy/6, extend recent observations that

this receptor type can be expressed by both thymic and

peripheral blood-derived Tcells [36, 461. Tw

o clones (JH6a

and JH8) w

ere isolated that express a TcR

yl6 unreactive w

ith theTiyA

, 6-TCS1 and B

B3 m

Ab (Table 1). B

iochem-

ical analysis showed that this T

cRy/6 contains no disulfide

bridge, that the y chain is approximately 55 kD

a and the 6 chain

approximately 40 kD

a (Figs. 2 and

3 and

not show

n). Previous studies have indicated that the 6-TCS1

antibody might be specific for V

bl[36] whereas B

B3 w

ould detect the V

63 gene product [37]. How

ever, Northern blot

analysis indicated Vbl

mR

NA

expression

in both the

6-TCS1- clones JH

6a and JH8, and in the 6-TC

S1+ clones JH

2a and JH12 (Fig. 4) but not in the control clone R

dVw

4 (G

-TC

Sl-BB

3+). Subsequent

sequence analysis

of the

cDN

A clones corresponding to m

RN

A encoding the 6

Eur. J. Imm

unol. 1989. 19: 2099-2105

chains expressed by JH6a, JH

8 and JH12 confirm

ed that these clones express aV

h1 gene product (Fig. 5). How

ever, for clones JH

6a and

JH8 an

identical sequence

was

obtained suggesting that these clones might be subclones

originating from one precursor cell. D

ifferences in the joining sequences betw

een the clones derived from JH

2a (6-T

CS1+), JH

6a (b-TC

Sl-) and JH

12 (6-TCS1+) w

ere evident (Fig. 5) supporting the biochem

ical data suggesting that these are independently derived clones (Figs. 2 and 3). T

he only clear difference between the 6 chain cD

NA

clones coding for the h-T

CSl+ receptors (JH

2a, JH12) and the 6

chain cDN

A clones coding for the 6-TC

S1- receptors

(JH6a, JH

8) is the use of the Jb3 gene segment in the latter

whereas the form

er use either the Jbl or Jh2 gene segments

(Fig. 5).

Using biochem

ical techniques we have not been able to

detect differences between the y chains expressed by the

6-TCS1+ clone JH

2a and the 6-TCS1-

clone JH8 (not

shown). A

lthough no formal proof, this supports the notion

that it is not a conformational determ

inant created by the com

bination of certain y and 6 chains that is recognized by the 6-TC

SI mA

b but rather a (conformational) determ

i- nant on the 6 chain itself. H

owever, the generation of

additional TiyA-G

-TCSl-B

B3-

clones expressing the V;,1

gene segment w

ill be necessary to evaluate the importance

of the 563 gene segment in determ

ining the lack of reactivity of the 6-TC

S1 mA

b.

In summ

ary, our results indicate that peripheral blood- derived TcR

y/6+ cells are heterogeneous both with regard

to CD

antigen expression and TcR

y/6 repertoire. Both

combinatorial and junctional diversification of the TcR

y/6 repertoire

has been demonstrated

using cloned T cells

derived from a single individual. A

lso we have character-

ized clones that express the Vbl gene segm

ent but lack reactivity w

ith the 6-TCS1 m

Ab indicating that this anti-

body is not an absolute marker for the expression of this 6

chain V region.

The authors would like to thank W

im Van Schooten, John H

aanen, A

nneke Jatison and Dietitie Bontrop for valuable advice during the

isolaiioti of the T cell clone5. and Drs. J. Bor.st. 7: H

ercend, M.

Brenner, L

. M

oretta and J.

E.

Coligan for the C

D36 and

EK ylb-specific antibodies.

Received June 21. 1989: in revised form

August 30, 1989.

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