Microbiol. Immunol. Vol. 23 (4), 223-232, 1979

Detection of Human B Cells and Chronic Myelocytic Leukemic Cells by Rosette Formation with Sheep Erythrocytes and Fresh Human Sera Po-Min

CHEN,*1 Noboru YAMANAKA, Kokichi KIKUCHI, Koichi KITAJIMA, and Ikuro KIMURA

Departmentof Pathology, Sapporo Medical College,Sapporo, and *SecondDepartmentof Internal Medicine, Okayama UniversityMedical School, Okayama (Received for publication,

May 24, 1978)

Abstract A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized withfresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34-58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.

Receptors and different antigens have been identified in lymphocytes, monocytes and granulocytes of all investigated mammalian species, including man (26, 29). For instance, B lymphocytes derived from bone marrow can be identified by the presence of surface immunoglobulin (SIg) which has the ability to bind specifically to antigenic determinates (18, 19, 24, 28). Receptors of the antigen-antibody-complement complex can be detected by rosette formation with erythrocytes sensitized with antibody and complement (2, 20, 27) and receptors for the IgG portion can be detected with soluble antigen-antibody complex or by fluorescein-labeled aggregated IgG (1, 3). Monocytes and granulocytes also have receptors for the activated third component of the complement (6, 8, 23). On the other hand, human thymus-dependent (T) lymphocytes form rosettes with untreated SRBC (E) (10, 14), and activated T cells bind with antigen-antibody complexes (Fc) (30). 1 Visiting

Research Fellow , Department of Pathology, Sapporo Medical College, Sapporo. Abbreviations used in this paper: SRBC, sheep erythrocytes; FHS, fresh human serum; PBL, peripheral blood lymphocytes; ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myelocytic leukemia; E, sheep erythrocytes; EAC, erythrocyteantibody-complement complex; T, thymus-derived; B, bone marrow-derived; GVB, gelatinveronal-buffered saline; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline.

223

224

P.-M.

CHEN

ET AL

In this paper, we report on the specific rosette reaction of FHS-coated SRBC (FHS-rosette formation) to human B and CML cells. The resulting procedure appears useful for detecting complement receptors of B and CML cells. METERIALS

Cell

suspension.

tonsils

during

phoma

were

clumps

for at

250

three

separated

by

through Dead 30

of

rosettes

E

PBL

were

of

in

washed live

three

than T

in

95%

of

CML high

cells

and

30-60

washed

and

who

showed

from

heparinized

eight

were of

the

sensitization,

cells

venous

blood

of rosette

formation.

exclusion

patient

with

test.

to

was

CML

who

showed

more blood

sediment

than

collected

spontaneously

collected,

last

infectious (5).

containing venous

allowed

the

with method

104,

heparinized

then

ALL

ALL)

(one

1.5 •~

C for

in

104

patients

who

with

and

washing,

(two

showed

T

WBC

by

30 were

Alsver's

washed

three

packed

cells

a

which

SRBC

untreated

patients

blue

were

percentage

cells

were

erythrocytes

were

procedure.

B cell

than

suspended

SRBC

37

six with

CLL

more

Indicator

FHS

one

a

patients

Before

cells

The

filtration

104-20 •~

from

solution.

gradient

The

tubes.

and

supernatant

shock

from

and

(6 •~

differential

trypan

column

relapsed

were

sedimen-

cell

in

over

CLL

and

peripheral

differential

with 3 •~

cell

WBC

seven

blood

gradient

T

104

ALL, in

with were

three

peripheral

B cell also

sedimentation

CLL)

separated in

a

Ficoll-

gradient.

blood,

layer

PBS

the

subject

or

RBC

gradient by

dye.

wool

separated

The

by

blood

leukocyte-rich

hypotonic

cells

ALL

blood

C.

in

the

Leukemic

Conray

37

times by

null

were

glasses,

sedimentation

subjects.

glass

the

normal

by

normal

determined

untreated

out

Cell

slide

purified

blood

11

siliconized

nylon

peripheral

The

three

the

of

a Ficoll-Conray

partially

venous

in

lym-

(5).

excluded a

by

were

from

was

from

eight in

at

min.

eliminated

with

PBL

granulocytes)

maintained

for

tonsils

pieces

carrying on

from

malignant medium.

two

and

from

cold

usually

purified

count

mature

kept

cells

from

WBC

gauze centrifugation

gradient

suspension

of were

cotton after

surgery,

with

RPMI-1640

between

heparinized

and

the

lymphocytes

mononucleosis

40%

times

organs

cardiac

patients

in

gradient

from

during of

suspended

Ficoll-Conray

Ficoll-Conray

lymphocytes

More

a

a

nodes

of

removed

separated

sedimentation then

a

thymuses

the

B lymphocytes in

from

and

sheets

METHODS

lymph

squeezing

were

min.

from

times

four

cells

g for

tation

or

washed

them

5 min.

obtained

tonsilectomy

were

filtering

Cells

AND

was min,

1%

the

degree

(sensitized

a final

1%suspension

From SRBC) in

in

were diluent.

PBS

of

10

in

use.

PBS 1:

was

noted the and

and In

from

heparinized

used

within

each

10

were

mixed

with

an

to

1: 80.

After

and

diluted

doses

remaining washed

50-90% three

venous one

washing

leukocytes was

from

doses,

removed

4 C,

sheep

hemolysis these

separated

at

before

SRBC

units of

were

stored

contained of

in

obtained.

SRBC

times which

suspension

diluted

SRBC

solution,

times

month.

the

upper

discarded. equal

For volume

of

maintenance of lysed of in

at 10-50%

non-hemolytic PBS

to

form

FRESH

Sera. from

FHS

were

rabbits,

and

anti-C3

werk

Ag,

Rosette

least

E

ice

for

cytes

37

to

(1 : 100)

was

twice

and

diluent

and

FHS,

to the

of

detct

rosette

Gel

(11).

con,

U.S.A.),

used

for

the

block

content

of was

before

the

kidney

or

each

a

5 min the

a

light

consisting

of

with 37

and

an

at

equal

C for

15

min.

reincubated

percentage

of

E

a

1%

of

on lympho-

37

C

1 : 5

30

serum Paul

in

mouse

(EAC)

min.

comple-

were of

The

EDTA-GVB,

washed

lymphocyte

percentage

PBS

and

formation.

of

normal

EDTA-GVB

(21). Bunnel

rosette

at

prepared

(9).

GVB,

of

diluted

milliliter

for

incubated

was

SRBC

calculated

or

sub-agglutinating E were

suspension

and

test

boiled

and to

FHS-rosette

ox

as

for

red

antibody 30

activity

min

blood

cut

1 cm

volume rosette

PBS

elution

collected

C,

with for

in

cells,

a

1:

and

GVB,ƒÊ=0.05,

for

18-20

1 cm2 hr.

of

20

cells

dilution

then

throughout or

formation

barbital activity

its

(12,

showed

three

Minicon-B of

used

to

the length. Each 13).

FHS

G-

elsewhere

major

rosette was

8.7.

and

and

well

Each

formation. isolated

FHS 23

cm

electrophoretic

eluate

Sephadex

detail

concentrators(Ami-

pH area

buffer.

with in

antibody

sectional After

packed described

detection

using a 5 •~

as

pattern

natural

segments of

column,

10

electrophoresis,

with

applied

long at

concentrated

was

zone

12 mAw

cm 6.4

The

fraction

block,

100 PH

was

electrophoresis

starch

into

to M PBS,

fraction

zone

FHS

at was

Forssman

fractionated.

starch

a constant and

applied

Each and

starch

pig

0.15

was

potato

by

with

was

FHS

FHS

current

absorbed guinea

EAC

cations

of

for

One-tenth

detecting

divalent

at

Sensitized

formation.

for

possibility

and

min.

SRBC

FHS-rosette

mixed

antiserum

suspension

30

of

was

suspension rabbit

this

ml

g

under

as

incubated

and

concentration.

three

absorb

with

peaks.

For

of

0.1

than

100

counted

and

quardruplicated.

106,

and

shaking

formation.

filtration

The

0.1%

twice,

C for

1%

2 x

0.2 ml) of mixed and

calculated.

a

washed

diluents

boiled

equilibrated

separated

a

of

serum at

was

of

at 37

with

were

suspension

200

volume

for

ml

calf

sensitive

equal

as

out both

FHS

Behring-

Laboratory,

gentle

defined

or

resuspended

E

were

was

duplicated

0.1

carefully

were

factors

used

rule

and

Cordis

by

cells

rosette

centrifugated

SRBC

more

was

at

three

to

used

was

obtained antiserum

(2 •~ 106, ml) were

resuspended

A

fetal

volumes

mixed

with of serum

To

than

from

volumes (1 %, 0.2

forming

test

25%

was

again

were

200.

2-mercaptoethanol

was

were

Each

was

resuspended

Test

were

U.S.A.

purchased

: Equal red cells

Rosette

suspension

Equal

incubated

bound

saline

:

min.

suspension

sera Anti-IgM

Laboratory,

was

cells

of •~

pellet

An

ment

The

with

more

of

RPMI-1640.

cells

cell PBS

The

30

min.

cells.

The

rosettes

C for

Fresh

immediately.

Miles

rosette formation and indicator

suspension

1 hr. binding

dose

225

U.S.A.

preparation.

E in

cell

EAC

30

red

:

1%

mixed

from

FORMATION

donors.

used

complement

FHS cells

C for

bound

of

normal

and

Wisc.,

a magnifications

rosettes

volume

pig

Gibco.,

a wet at

three

The

37

into

microscope

19

mice,

purchased

Guinea and

at

pipetted

were

.formation. or tumor

incubated

and

ROSETTE

Germany.

Florida,

lymphocytes

from

pigs

antiserum

Complement. Miami,

SERUM

obtained

guinea

West

HUMAN

at length,

run, segment was

from 2 ml

tested

the was for

was

and starch eluted protein

a

P.-M.

226

Ouchterlony 1.2%

agar For

200

gel

and

in

anti-IgM

stand

zone

at

ET AL

immunoelectrophoresis pH

absorption

starch

forming

and

buffer, ƒÊ=0.05,

antiserum or

to

rosette

precipitation

barbital

filtration

allowed

detect

gel

gel

CHEN

8.7

tests,

fractionated

were

temperature

for

30

were

performed

with

22).

samples

electrophoresis

room

(4,

added

min.

from to

The

FHS

anti-IgM samples

by

G-

antiserum were

used

to

activity.

RESULTS

FHS

Rosette Various

formation forming of

cells

lymphoblasts

Formation

using

and

FHS,

rosettes. in

with

normal

E

Lymphocytes neoplastic and

EACm.

Proportional

normal in

Table

Table

subjects, B

1.

2.

cell

and

rates 71.3•}2.5%

ALL

(7)

Lymphoblasts

lymphoid

and

or

Tables for

FHS of

myeloid 1 and

B

rosette cells

75.8•}5.9%

Percentage

of B cells

forming

Percentages

of T cells

forming

cells 2 show formation

separated of

rosettes

rosettes

were the

with

with

were from

cells

in

tested

for

percentage

tonsils,

FHS-coated

SRBC

SRBC

cells

21.5•}2.5%

B lymphomas.

FHS-coated

rosette of

68.5%

of Less

FRESH

HUMAN

SERUM

ROSETTE

FORMATION

227

than 7% of T cells separated from PBL and less than 16% of cells in T or null cell ALL were positive in the same tests (Table 2). Thymocytes, on the other hand, showed 42% rosette forming cells at 37 C incubation because some thymocytes formed rosettes with the E receptor on indicator red cells, as in EAC (16). FHS RosetteFormationwith LymphoidCells in CLL FHS-rosette formation activity was shown in human B lymphocytes and B lymphoblasts, but not in T cells or null cells of PBL, tonsil, leukemic and lymphoma cells. FHS rosette formation was not found in B-CLL cells, except for one case (Table 3). Less than 8% of cells formed rosettes in seven out of eight cases of T and B cell CLL. In contrast, 26-70% of EAC rosette forming cells were positive in B-

Table

3.

Percentages

of CLL

cells

forming

rosettes

with

FHS-coated

SRBC

Fig. 1. rosette higher SRBC tion.

A close correlation was evident between FHSformation and maturation of CML cells. A rate of rosette formation and more adhering were found with greater CML cell matura-

P.-M.

228 CLL.

Cells

C3d

receptor-positive,

FHS

Rosette

tion

of

binding of

adhering

one

Close

CML

cells or

of red

CML

to

1).

cell

at

Less

(Table

than

but

the

membrane

forming 1,

ET AL

showed

60%

FHS-rosette

formation

were

later.

Cells

were

SRBC,

which

discussed

rosettes

(Fig. two

rosette cells

be

correlations

binding

72%

case

will

with

formed

1).

of one

B-CLL as

cells

SRBC

than

the

Formation

CML (Table

of

CHEN

a

rate shown 1% percentage surface

polymorphonuclear

of

41.2•}8.5%

between

with rosette

of myeloblasts

and

of rosette increased

FHS-coated

formation promyelocytes

formation with

leukocytes

cell

and

diluted

Fig. 3. Percentages of FHS-rosette formation of PBL, tonsil cells, B lymphoma and CML cells are presented. (a) About 80% cells of B lymphoma or CML cells formed rosettes with FHS-coated SRBC. (b) But the activities were abolished, when FHS was inactivated with heat at 56 °C for 30 min. (c) Rosette formation reappeared after addition of partially hemolytic guinea pig complement.

maturashowed

the

maturation. were

Fig.1).

Fig. 2. Addition of partially hemolytically FHS proved optimal for rosette formation.

SRBC

and

surrounded

number More by

FRESH Table

4.

HUMAN

Rosette

formation

SERUM

ROSETTE

FORMATION

percentages

in B cells

human

animal

and

and

CML

229 cells

with

sera

QuantitativeEffectsof Serum The concentration of FHS used for SRBC coating also affected the results. The upper limit of serum concentration was set by requirements for partial hemolysis of red cells. Figure 2 shows the effects of several serum concentrations on rosette formation of B cells and CML cells. From these experiments, the number of rosettes formed was shown to be in direct proportion to the amount of FHS on red cells. A high percentage of rosettes was present in partially hemolytically diluted serum and at lower concentrations, a decline in rosette formation was observed. SRBC sensitized with heat inactivated human serum did not form rosettes with B and CML cells. However, when a partially hemolytic dose of guinea pig complement was added, rosettes were formed (Fig. 3). This suggests the necessity of complement in FHS rosette formation. RosetteFormationwith SRBC Coatedwith Sera of OtherAnimal Species SRBC coated with FHS and with fresh rabbit or guinea pig serum produced rosettes with B and CML cells. An interesting finding was that SRBC sensitized with undiluted mouse serum formed rosettes with B cells but not with CML cells even when undiluted serum was used (Table 4). However, CML cell rosette formation was restored when a partially hemolytic dose of guinea pig complement was added. Analysisof SerumFactors,for FHS Rosettes ED TA-GVB absorptiontest. In the absence of divalent cations (EDTA-GVB), lymphocytes did not form rosettes with FHS-coated SRBC, but in GVB, PBS or normal saline diluent, lymphocytes formed rosettes as in EAC receptors. Davidsohnabsorptiontest. There was no change in the percentage of FHS rosette forming cells when either B cells or FHS were absorbed by a suspension of boiled guinea pig kidney or boiled ox red cells.

230

P.-M.

CHEN

Fig. 4. FHS-rosette formation activity was found in Fl fraction of fresh human serum fractionated with Sephadex G-200.

ET AL

Fig. 5. fresh

FHS-rosette formation activity of human serum components frac-

tionated with starch zone The activity was presented tube

No.

electrophoresis. in fractions

of

6-10.

RosetteFormation in Fractions of G-200 Gel Filtration and Starch Zone Electrophoresis Each fraction obtained from G-200 gel filtration or starch zone electrophoresis was tested for rosette formation. Activation was present at Fr. 1 in G-200 gel filtration (Fig. 4) and tube no. 6-10 in starch zone electrophoresis (Fig. 5). These fractions were found to contain IgM in examinations by Ouchterlony gel precipitation and immunoelectrophoresis. FHS rosettes were inhibited when these fractions were absorbed by anti-IgM antiserum. DISCUSSION

Receptors for fixed C3 of complement are known to exist on the surface of human monocytes (8), neutrophils (6, 15, 23) and lymphocytes (17, 27) due to rosette reaction in the presence of red cells sensitized with antibody and complement. AntiRBC rabbit antiserum was required as antibody in EAC rosette formation. This study presented evidence that fractions of normal PBL, tonsil cells, B lymphoblasts in ALL and lymphoma, and granulocytes in CML have receptors for FHS-coated SRBC. Lymphocytes, lymphoblasts and CML cells mixed with SRBC sensitized by FHS formed rosettes, but rosette formation did not occur with SRBC sensitized by heat inactivated-serum. Thus, it was suggested that natural antibody and complement might be involved because the antigen-antibody-complement complex is known to adhere to B cells in classic EAC rosette formation. This kind of natural antibody to SRBC exists in human and animal sera. The percentage of rosette forming cells in this method was the same as that for EAC. FHS-coated SRBC had a high affinity to B and CML cells when mixed together or without packing by centrifugation, and FHS-rosettes formed within 10 min at 37 C. The conditions of rosette formation reported here were optimal and stable in the presence of partially hemolysized, diluted serum for sensitization of SRBC. The reasons why indicator red cells react with B cells may be as follows: (a) Percentages of rosette forming cells reacting in this method plus percentages of rosette

FRESH

HUMAN

SERUM

ROSETTE

FORMATION

231

forming cells reacting in E rosette formation were nearly 90-100% in many cases (Table 1). The number of rosette forming cells as estimated by FHS-rosettes approximated the number of EAC positive cells. (13)Sensitized SRBC reacted with B cells separated from tonsils and with lymphoblasts in B cell ALL and B cell lymphoma which contained 90-99% SIg positive cells, but did not react with T cells in PBL, lymphoid cells in T cell CLL, lymphoblasts in T cell lymphoma and T or null cell ALL. (c) It was demonstrated that FHS rosette formation was complement dependent and was inhibited by anti-C3 antiserum. The serum factor for rosette formation contained IgM. One unresolved question was why the optimally diluted serum lysed 10-50% SRBC. The remaining 50-90% SRBC developed rosettes. However, no rosette formation was shown at nonhemolytic doses. This suggests that hemolysin in serum might play a role in FHS rosette formation. In our data, unlike in EACm rosette formation, most CLL cells were not detected with FHS-coated SRBC, and many CML cells were detected with FHS-coated SRBC. A similar observation was made by Michlymer and Ross (17, 25), who reported that EAC (antiserum) prepared with humen complement reacted poorly with CLL lymphocytes. Normal lymphocytes have C3b and C3d receptors, but most cells contain only the C3d receptor. EAC prepared with a high dilution of FHS did not contain C3d to detect the C3d receptors of CLL lymphocytes. Granulocytes, on the other hand, were found to have C3b receptors but not the receptors for C3d. Our FHS rosette formation most likely detects the C3b receptors. In conclusion, FHS rosette formation may be used to detect EAC rosette formation. This is a new and convenient method for detecting C3b receptors. In the test system, antiserum is not required. The same method may be applied to detect C3d receptors when fresh mouse serum is used instead of fresh human serum. REFERENCES

1) Basten, A., Miller, J.F.A.P., Sprent, J., and Pye, J. 1972. A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J. Exp. Med. 135: 610-626. 2) Bianco, C., Patrick, R., and Nussenzweig, V. 1970. A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. Exp. Med. 132: 702-720. 3) Brown, J.C., de Jesus, D.G., and Holborow, E.J. 1970. Lymphocyte-medicated transport of aggregated human y-globulin into germinal center areas of normal mouse spleen. Nature (Lond) 228: 367-369. 4) Grabar, P. 1960. Use of immunoelectrophoretic analysis in the study of specific precipitation, p. 21-29. In Heidelberger, M., and Plescia, O. J. (eds), Immunochemical approaches to problems in microbiology, Inst. of Microbiology, Rutgers, U.S.A. 5) Greaves, M.F., and Brown, G. 1974. Purification of human T and B lymphocytes. J. Immunol. 112: 420-423. 6) Henson, P.M. 1969. The adherence of leukocytes and platelets induced by fixed IgG antibody and complement. Immunology (Lond) 16: 107-121. 7) Hiraki, S., Miyoshi, I., Masuji, I., Kubonishi, I., Matsuda, Y., Nakayama, T., Kishimoto, H., Chen, P., and Kimura, I. 1977. Establishment of an Epstein-Barr virus-determined nuclear antigen-negative humen B-cell line from acute lymphoblastic leukemia : brief communication. J. Natl. Cancer Inst. 59: 93-94.

P.-M.

232 8)

Huber,

H.,

Two

9)

Polley,

distinct

M.J.,

receptor

162:

1281-1283.

Ishii,

Y.,

Koshiba,

H.,

Characterization 10)

Jondal, I.

Linscolt, sites

for

J.,

Holm,

A large

G.,

H., Wigzell,

H.H.,

component

Maeyama,

I.,

Takami,

1972.

T.,

Mueller-Eberband, and

Ishibashi,

antigen.

Surface

forming

and

of complement

specific

H.

of lymphocytes

ET AL

Findenberg,

third

B lymphocyte

and

population

W.D., the

Ueno,

of human.

CHEN

J.

F.,

on

Science

K.

1975.

466-469.

T

with

1968.

globulin.

Kikuchi,

114:

human

rosette

H.J. G

and

Immunol.

marker

non-immune

for

and

SRBC.

B lymphocytes.

J.

Exp.

Med.

136:

207-215. 11)

Killander, phoresis.

12)

Koyama, cells.

13)

14)

15)

16)

J.

1963.

Acta

Soc.

J.,

and

Japan.

Kunkel, Exp.

Lay,

Exp.

W.H.,

Lay,

W.H.,

128:

991-1007.

Mendes,

N.F.,

Tolnai,

aspects

G.,

lymphocytes. 18)

Miller,

for

Nossal,

radio

and

on

in

1968.

tests

filtration

rabbit

a starch

and

zone

electro-

hemolysin

to sheep

red

supporting

medium.

Proc.

J.

H.

1970.

105: J.,

Warner,

complement

on

Gilbertsern,

detect

R.B.,

human

Immunol.

of sheep

red

sites

leukocytes.

and

J.

cells

to

Exp.

Metzgar,

complement

111:

Receptor

Binding

a

Med.

R.S.

receptor

(B)

1973.

and

sheep

860-867.

for

complement

on

certain

human

blood

670-676.

Basten,

A.,

Nature

and

(New

N.L.,

immunolabelling

1971.

531-532.

for

N.P.A., to

V.

230:

Receptors

used

lymphocytes.

Huber,

Sprent,

Nussenzweig, Nature

Silveira,

rosette

B lymphocytes.

G.J.V.,

wich

V.

Immunol.

J.F.A.P.,

serum 19)

J.

C.,

M.E.A.,

and

gel

studies

electrophoresis

lymphocytes.

(T)

Michlymer,

Immunochemical

Zone

Bianco,

of the

erythrocyte-binding 17)

1959.

1952.

Nussenzweig,

N.F.,

Technical

by

42-44.

of human and

immunoglobulins

230-244.

551-559.

R.

80:

Mendes,

population

29:

Slater,

Med.

68:

H.

Med.

and

Biol.

large

of human

Upsil.

Nashimoto,

J. H.G.,

Soc.

Separation Med.

Warner,

Biol.)

Lewis,

H.,

technique

and

for

N.L.

237:

1972.

Selective

cytoxicity

of anti-kappa

18-20.

Sprent,

J.

lymphocyte

1972.

Quantitative

surface

receptors.

features J.

Exp.

of

Med.

a sand-

135:

405-

428. 20)

Nussenzweig, Possible demic

21)

22)

23)

24)

V., role

Press, H.,

human

origin

O.

chemical

approaches

Rabellino,

E.M.,

eosinophil

Raff,

M.C.,

Ross,

gel

in

Receptors

Ltd.

In

for

Progress

C3 in

on

B lymphocytes;

Immunology,

Phillips,

J.M. Ann. H.M.,

Grey, surface

Aca-

and blood

T.O.,

Exp.

and

Nossal, and and on

Y.

in

In

vitro

E.,

chronic

surface

B. of

1972.

(eds),

by

Immuno-

U.S.A.

macrophage,

cells

1973.

neutrophil

in

the

peripheral

Tissue

localization Immunol.

and

marrow Exp.

leukemia.

mouse

J.

P.,

bone

Evidence

different

complement

of two 108:

and

populations

1456-1460.

lymphocytes

by

anti-

793-798.

Campbell, The

Two

of phagocytes

129:

J.

activated

H.M.

1972.

Identification

lymphocytic

on

of

688-692.

receptor.

immunoglobulin. 1971.

obtained

O. J. Rutgers,

IgG

cells

798-811.

sensitization Sci.

II.

and

cultured 521-522.

patterns

Plescia,

of Į-bearing

Grey, 138:

M.E.A.

Acad.

C3 115:

membrane

Rabellino,

G.J.V.

Andersson, the

and

Tolnai,

1966. N.

precipitation and

dependence

Med.

by

248:

931-942.

E.M., J.

of lymphocytes.

surface-bound

J.D.

18:

on

Nature

of Microbiology,

for

Immunol.

Thymus

distinguished

complex.

Inst.

J.

erythrocytes

M.,

Receptor

vitro.

Rabellino,

human

membrane. immune

microbiology,

1970.

N.F.,

cell

Heidelberger,

1975. in

Immunology

Mendes,

detectable

in

H.H.

M.J.,

of

by

In

D.

Wortis,

E.R.,

401.

1971. B.

of comparative

of mice.

on

1:

A.

Amos.

formation

5-19.

growth

lymphocytes.

the

Rosette

problems

Polley,

and

immunoglobulin

p.

cell

globulins

Yoshida,

Eden,

73.

of complement

Metcalf,

lymphocytes

peripheral 30)

to

Unanue,

Wilson,

1974.

human

N.P.A.,

J.W.,

with 29)

and p.

Interpretation

and

and

gen-antibody 28)

T.

colony

G.D.,

Uhr,

P.,

response,

techniques,

tissues

Silveira,

Dukor,

activation 1960.

in

and

C.,

immune

Baba,

and

diffusion

of human 27)

and

Ouchterlony,

receptors 26)

the

N.Y.

Okada,

lymphoid 25)

Bianco,

in

for

Med.

Schmidtke,

as the 133:

of human

T

Lancet

2:

a receptor thymus

main

J.

1971.

source

Immuno-

of lymphocytes

1188-1198. and

B lymphocytes

in

normal

788-791. recognising

lymphocytes.

antigen Scand.

complexed J.

Immunol.