THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1976 by The

ANt)

QUANTITATION

Vol.

CYTOCHEMISTRY

Histochemical

Society.

OF

24, No.

1, pp. 378-382, 1976 Printedin U.S.A.

Inc.

LYMPHOCYTE

RESPONSE

TO

ANTIGEN

BY

FLOW

CYTOFLUOROMETRY’ J. D. BRAUNSTEIN, Sloan-Kettering

R. A. GOOD,

Institute

for

J. A. HANSEN.

Cancer

Research,

and

York,

New

T. K. Memorial

New

SHARPLESS Hospital

York

M.

R.

and

Allied

AND

for

Cancer

MELAMED Diseases,

10021

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated “pyroninophillic” immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14G.thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine

incorporation

was

response secondary fluorometry. Kinetic compared to normal AO cytofluorometry lymphocyte proliferative

Lymphocytes genic and

by

the

cellular

has

mitoses

(8),

to

respond

vitro

by

proliferation

production

of protein (9).

been by

showing

nibonucleic

lapse

(DNA) (12)

ninophillic nibonucleic

immunoblasts acid (RNA)

mediators counting

(6),

deoxy-

3H-thymidine

and

‘4C-thymidine by counting

(4). content

The of

and pyro-

a prior

technique formed

for cells

flow

cytofluomometry

ing

(1).

guished

from

creased

RNA

The proliferative lymphocytes was number

(per

diseases while

(5).

It has DNA

which

causes

B them

blast cells lymphocytes

was aided

by the

has

presented

demonstrated

synthesis

stimulating

antibody-secreting ating (7). Similarly, work

been

This immunodefiwith

to

following

with

develop

into

cultures bacterial

proliferbe stimugrants

(per

from

the

U.S. Public Health Service: Grants CA1740401 and CA05826 from the National Cancer Institute, Grant AI11843 from the National Institute for Allergy and Infectious Diseases, and Grant 126CA14134 from the National Bladder Cancer Project, the Bauman Fund

and

the

American

Cancer

cent

cells for each

indicated

by the

of values of

per

immunoresponsive

with

time.

cell

(syn-

increased could responding

varying

be

with

In this paper this technique

we to

lymphocytes stimulated with The proliferative response

immunoresponsive

the

in-

for a population of in an increase in the

stimulation. application

and

their

cells).

per cell. The batter a mean value for the

of human antigen.

distin-

by

response was

stain-

were

of responsive

or as a distribution

time following describe the

hydrox-

lymphocytes without can

cells

in

as

orange

cells

a

transusing

(pyroninophillic

response reflected

cent)

content

certain

acridine cells

content

RNA

dissociated. in

we described and counting per cell RNA

and

increased pyronino-

factor,

phytohemagglutinin-

nonresponsive

of protein)

sometimes

with

lymphocytes,

Immunoresponsive

thesis

clinically

inhibitory

identifying with increased

immune

are

immune

(11).

study human

nonproliferative

and

depressed

migration

stimulated

ive

by inhibiting

Strategy

produce proliferation

In

of re-

formed

to

without

nonproliferative

vitro

This

and

bated

(3)

The

ciency

1

with

of active response.

seen

mitogen,

patients

is also evidence a nonprobifemative

responses

yurea

from

blast cells production,

Proliferat been

or (3)

Cells

to anti-

by

with

counting

7.

proliferative

in newly

radioautography scintillation

day

cinematography

increase

acid

phillic protein

The

demonstrated time

an

on

known in

immunity

sponse by

are

stimulation

seen

to cancer showed bower than normal antigen response by cytostudies revealed both a lower percentage of immunoblasts when and a lower average per cell RNA content of the stimulated cells. is suggested as a convenient method of simultaneously assessing and nonproliferative response to antigen.

nonprobiferative

cells response cell)

with (RNA

are

time) content

measured

si-

multaneousby. MATERIALS

in

Society.

Lymphocyte culture: hepanin (20 units/mb)

378

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on March 15, 2015

AND

METHODS

Venous blood and diluted

was

with

collected

Hanks’

QUANTITATION CANDIDA 14c 16

ANTIGEN

Thymidin.

Normal

0’

1:10 - -

0

8

1:30

0

lllOO no

RESPONSE

STIMULATION

Blood

antigin

‘0

.-.-----.

LYMPHOCYTE

dilution

/

.1

tion

‘of 4/

0

1

1

Each

for

,,

12

I

us

control

Data

are

assay:

I

U

I

onto

water.

with

a

After

scintilla-

expressed

as average cultures

are

stimulated

Cultures

for cytofluorom-

STIMULATION

ANTIGEN

Cytolluorim.Iric

.

aspirated

cultures).

CANDIDA So

Fi&wosarcoma

was

analysis were aspirated from each well and the were washed with phosphate-buffered saline at pH 7.4 (PBS). They were then centrifuged for 10 mm at 170 x g, the supernatant was removed and the cells were resuspended by vortexing in 0.1 ml PBS. After

“4

0

be-

etric cells

0

12

culture

counting.

Cytofluorometnic

1

Normal

i

machine.

fluid

and

‘O

hours

cpm of triplicate cultures. Stimulated measured in net cpm (difference between

4

,

Twenty-four

glass fiber filter and washed with distilled drying, the filters were placed in vials

I’

antig.n

0

incorporation:

tisample

I’

50

379

ANTIGEN

fore harvesting, 25 zl of “C-thymidine (60 mC/mM, 2 pC/ml) in culture medium were added to each cell culture. The cultures were harvested using a mul-

Lymphocytes

“

TO

Thymidine

Incorporation

Periph.ral

.---

12

OF

Normal

Ouanlitotion Psriph.ral

of

Slood

Imn,unoblasts

Lymphocyt.s

U

patisnt

110

#{176}

40

0

I

- -

a

afligIn

dilution

1:30

“

IS

#{149} 1:100

“

I’

30 nO

0-#{149}

onligsn

20 4

,

I,

.

.

,

2

3

Days

1. Dose

FIG.

corporation of peripheral

C:---’ S

4

7

6

10

Incubation

response

curves

of

“C-thymidine

in-

into Candida antigen-stimulated blood lymphocytes

cult unes .

Normal

40

0

balanced salt solution (Ca Mononuclear cells, isolated g for

30

were

washed

Roswell taming

mm

on

adjusted

Institute

streptomycin human

count

triplicate

cells

medium (1.2 The

with of the

size

(HBSS). at 330 x gradient,

resuspended

in

1640 mM),

(10 units/mb)

serum.

determined

cytes. Cultures culture medium phytohemaggbutinin in

and

l-glutamine

was to

density

HBSS

Hepes,

normal

centration

in

Memorial

25 mM

bin (10 units/mb), pooled

a Ficoll-hypaque

twice

Park

and Mg free) by centnifugation

lymphocyte

a Coulter of small

con-

peniciland

round-bottomed

microtiter

to be

frozen

until

used.

:

:1’ 1

.!20

‘

I..

I 0

‘

.

10

15%

Counter

‘

, Flbronorcomo

40

,

,

,

,

,

polIsnI

lympho30

p

...,

plates

(Cooke Engineering, Alexandria, Va). They were incubated at 37#{176}C in a humidified atmosphere contaming 5% CO2 and then were prepared for cytofluorometnic assay or scintillation counting. Antigen: Candida antigen was prepared from cultures of washed Candida albicans by sonication, and the aqueous supemnatant was diluted in Roswell Park Memorial Institute medium 1640 at various concentmations,

/,#{149}#{149}‘d’”

con-

of 5 x 10’ lymphocytes in 150 tl of plus 25 l of Candida antigen, (PHA) or medium were prepared in

,30 -4

:: _______________

11

I

0 Doys

Incubotlon

FIG. 2. Dose response curves “pyroninophillic” blast cells stimulated cultures of peripheral

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on March 15, 2015

of per cent stimulated in Candida antigenblood lymphocytes

380 fixing for 3-24 ethanol:acetone, and

the

by the the samples

hr

supernatant

was

BRAUNSTEIN

ET

addition of 0.9 ml 1:1 were again centrifuged,

Figure

removed.

Each

sample

are

orange

metachromatically

(AO),

meric

form

RNA

to

which

stained

intercalates

to fluoresce

fluoresce

green

red

(2).

by

into ( 10) and

Each

was

is allowed

for

retrieval

and

display

at

a later

time.

The

cells

corresponding

the

In practice

control

the

threshold

was set such that approximately the control population were lated. ‘

little

dilutions

of

AO cytofluorometry b. This shows peak per

cent antigen

DNA

difference antigen.

is shown responses

stimulated concentrations

in on lym-

observed. patients carcinoma

ished

with

response

AO

Hodgkin’s fibrosarcoma,

and

to antigen

cytofluorometmy

corporation. patient with

was

and

COMPARISON OPTIMAL

OF PHA

OPTIMAL

RESPONSE

observed

by

Kinetic studies fibrosarcoma

pana diminboth

by

‘4C-thymidine in the (Figures

ANTIGEN OF

disease,

in-

case of the ic and 2c)

RESPONSE

NORMAL

WITH

LYMPHOCYTES

number

cells (immunoblasts) in each sample by counting the cells with red fluoresgreater than that of the unstimulated

in

Variations in at different

of the

7, with

different

day 6. phocytes

creatic

to

of stimulated was determined cence intensity samples.

from with 2a and

In

equilibrate for 1 mm and then is measured by means of a Cytofluorgraf 4801 (Bio/Physics Systems Inc.. Mahopac, N.Y.) interfaced to a Nova 1220 minicomputer (Data General, Southboro, Mass.). The red and green fluorescence intensities per cell are measured for populations of 5000 cells/culture, usually in 20-30 sec, and the data are recorded in computer memory

incorporation

on day

Kinetics Figure

were

acridine

b. Peak

occurs

resulting

DNA in monois ‘stacked” on

sample

la and

precursor

resuspended in 0.5 ml of 10 M ethylenediaminetetraacetate buffered to pH 6.0 with 10 M NaH2PO4, denaturing double-stranded RNA (2). The cells were then stained in suspension by the addition of0.5 ml 3 x 10 M AO in PBS. Under these conditions nucleic acids

AL.

(unstimulated)

for

red

97 defined

fluorescence

of the cells in as ‘unstimu-

RESULTS

The stimulated by

two

and dine phocytes

AO

proliferative in vitro methods,

response by antigen

of lymphocytes was quantitated

‘4C-thymidine

incorporation

cytofluorometry.

incorporation from

Kinetics

Unntimulated

FIG. 4. lymphocyte

of ‘4C-thymi-

into antigen stimulated bymnormal subjects is shown in

two

HISTOGRAMS

which

Comparison (red

result

from

STIMULATED

antigen

of

intensity)

RNA

of blast

stimulation

and

from

per cells

PHA

stimulation

OF PER CELL FLUORESCENCE

ANTIGEN

of distribution

fluorescence

NORMAL

INTENSITY

LYMPHOCYTES

III! .

:ii

I

Unutimulat.d

FIG. 3. peripheral

I_IIII day

Histograms blood

I

I

I

I

4

I

I

I

I

i_111_jL_.I I day

of red and

green

fluorescence

1

1

5

1

1

1

1

1 day

intensity

of Candida

-

L-.L._L_.L__L_L_1._1I-1:::7

6

day

antigen-stimulated

lymphocytes

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on March 15, 2015

7

cultures

of normal

QUANTITATION

OF

HISTOGRAMS STIMULATED

LYMPHOCYTE

RESPONSE

TO

OF PER CELL FLUORESCENCE OF LYMPHOCYTES FROM FIBROSARCOMA

381

ANTIGEN

ANTIGEN PATIENT

7

Unstimulated FIG.

5.

Histograms

lymphocytes showed

from that

was

this cells

(Figures

3 and

shows

per

cell

a variation

than

and

radioactive

cell

The

in

4).

We

cell

red

with

have

also

cells

compared at comparable

pop-

induced a higher

by RNA

with

induced Candida

antigen

found

a higher

percent-

in PHA-stimulated antigen-stimulated times.

Quantitation has

several

available.

advantages The

quantitating ative

both

Secondly, mm

remains

cub-

peripheral med for

yielding bias with

over

separately

and and

in cell

methods

cells in less

significant 50,000

is in

The cells,

than

studies

be 1

results test

can

a number

be

cell

to distinguish

similar weed

Whether or the responses

a

this

in

from as yet

differences mitogen

in and

not these differof the same cell

stimuli or to differences effector cell functions

few

normal

technique.

later

and

states

will to

paper2

we will reaction It

seems

carried

out

‘Braunstein JD, Melamed pont B, Good RA: Quantitation sponse in mixed lymphocyte fluorometry,

be

abnormal been and

manuscript

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on March 15, 2015

with alter

exammito-

values

reported

compared

known

lymphocyte already

and

have antigens Normal

in response

cytofluorometry.

can

poke

blood specimens responsiveness to

sponse. In a future mixed

simultaneously.

by cell

selection. of

only

by

disease

nonprolifer-

of 5-10,000 cell

statistically samples

other advantage

proliferative

measured

A. to

yet,

detail

by paper,

individual

to be seen.

As

response in this

important

populations and

without

used

most

responses,

counted

immune

as reported

of the

that yield different amounts We have shown differences

experiments, PHA and

to

variations

of cellular

cytofluorometry,

blood. Thirdly, working with

content between blast cells resulting and antigen stimulation and, in

cub-

gens

of

it possible

population to different which rebate to specific

DISCUSSION

AO

nature makes

concanavalin ences relate

by

this

blood

tracers. quantitative

unpublished response

opticon-

peripheral

from peripheral inconvenience

blast cell responses of RNA per cell. RNA PHA

fluorescence

do immunoblasts

of responsive

tures tures

normal

cell (Figure 3). This may antigens. We have found,

stimulation

(Figure

antigen-stimulated

3).

available avoids the

responsive

responding

that immunoblasts stimulation have

optimal age

the

content) per for different

tent

of

Figure

measurements

stimulation,

however, mal PHA

it

to a change in the percentage of in each culture as cells respond

to antigen (RNA differ

of

5).

In addition immunoblasts ubation

with

with

easily

percentage

per

intensity

(compare response

a lower

content

7

fluorescence

of proliferative both

RNA

green

fibrosarcoma

compared

average

day

and

with

failure with

responsive

red

a patient

associated

lower

of

and in more

findings

in

lymphocyte

report

a study

quantitated quite that MR,

of the by

likely other

re-

AO from

immuno-

Hansen JA, Duof lymphocyte meculture by flow cyto-

in preparation.

382

BRAUNSTEIN

logic mation

reactions involving can be quantitated

lymphocyte by this

LITERATURE 1.

Braunstein Traganos oftransformed

response.

antigen 132:327,

MR, Darzynkiewicz Good RA: Quantitation by flow cytofluonime-

Clin

Immunol

Z,

Immuno-

1975 Z, Traganos

F, Sharpless T, Melamed MR: Conformation of RNA in situ as studied by acnidine orange staining and automated cytofluorometry. Exp Cell Res, in press 3. Dutton RW and Eady JD: An in vitro system for the study of the mechanism of antigen stimulation in the secondary response. Immunology 7:40,

2.

4.

Darzynkiewicz

1964 Elves sponse

vitro. 5.

MW, Israels of lymphocytes Lancet 1:806,

Levin AS, Spitler Wiskott-Aldnich

MCG, to

Collinge antigen

M: The challenge

rein

LE,

Stites

DP,

Fudenbeng

a genetically

HH: deter-

stimulated 1969

lymphocytes.

J

Exp

Med

7. Melchers F, Andersson J: Proliferation and maturation of bone marrow derived lymphocytes, Cellular Selection and Regulation in the Immune Response. Edited by GM Edelman. Raven Press, 8.

9.

10. 11.

New York, 1974, p 228 Permain G, Lycette RR, Fitzgerald PH: Tuberculin induced mitosis in peripheral blood leukocytes. Lancet 1:637, 1963 Remold HG, Katz AB, Haber E, David JR: Studies on migration inhibitory factor (MIF). Cell Immunol 1:133, 1970 Rigler R: Binding of acnidine orange to nucleic acids. Acta Physiol Scand [Supplj 67:32, 1966 Rocklin RE: Production of migration inhibitory

factor

1963

Syndrome,

AL.

mined cellular immunologic deficiency. Proc Natl Acad Sci USA 67:821, 1970 6. Marshall WH, Valentine FT, Lawrence HS: Celbular Immunity in vitro: Clonal proliferation of

transfortechnique.

CITED

JD, Melamed F, Sharpless T, lymphocytes

try. I. PHA pathol 4:209,

ET

12.

by non-dividing

110:674, 1973 Schrek R: Cell duced in vitro.

lymphocytes.

transformations

Am

Rev Respir

Downloaded from jhc.sagepub.com at UNIV OF SASKATCHEWAN on March 15, 2015

and

J Immunol mitoses

Dis 87:734,

pro1963