Monocytes from mobilized stem cells inhibit T cell ... - Semantic Scholar

Monocytes

Abstract:

from mobilized

Kazuhiko

Ino, Rakesh

Department

of

Pathology

K. Singh, and James and

Microbiology,

Granulocyte-macrophage

factor, products,

mobilized and

contain

University

blood blood cells

stem cell leukocytes

that

cause

(PSC) post-

allogeneic

and autologous T cell apoptosis. Isolation and acterization ofthese cells demonstrated that they low-density T cells in

(Percoll fractionation) PSC products have

magglutinin rifled

(PHA)

CD4

normal inhibitory CD8 TCRa/

or

CD14’ a depressed

mitogenic CD8

response;

T cells

mitogenic activity

function. was observed

cell-depleted T cells.

fractions Inhibition

monocytes. phytohehowever,

exhibit

no CD4,

T cell and

in CD4CD8 cell function

by

CD14 monocytes required cell-cell contact, and the analyses of DNA fragmentation by Southern and TUNEL analysis demonstrates an activation-induced T cell apoptosis in the presence ofCDl4’ monocytes. Reverse-transcriptase studies suggested tumor

necrosis

ucts lion.

may

Key

Words:

J.

that factor

polymerase high levels gene

chain reaction of interleukin-lO or

transcripts

PSC

in the

contribute to the inhibition Leukoc. Riot. 61: 583-591;

of

Nebraska

of T cell 1997.

but

plantation

.

.

peripheral

blood

stem

cell

ablative

and

prolong

chemotherapy

or reinfusion

[8,

peripheral

blood

tance.

this

These

9].

(PB)

leukocytes

associated creased

observation

cells,

which

with by tumor

controversial

post-transplantation

more,

func-

proliferative pared with

cells

from

explanation

studies

and could

transplantation

with

MATERIALS

AND

depleted,

isolated

products

the

lineage remains that

CD4

and

a normal (PHA) comThis provides

dysfunction

that the immune

mobilized

function Further-

demonstrate

immune

suggests improve

are

can be inof hematopoi-

demonstrate

to phytohemagglutinin stem cell products.

for

transplantation cell products

and

can inhibit T cell T cell apoptosis.

are

PSC

response undepleted

impor-

function,

15]. However, the cellular T cell inhibitory activity These

CD14

T cells

clinical

T cell

hematopoiesis [12-22] secretion or administration

[12-22].

when

potential

inhibit

CD14 cells in PSC products and lead to activation-induced CD8

co’onyas well

products

functions [7, 10, 11]. of positive and nega-

has can

growth factors [14, mechanism of this

Thus,

and overcome imto reduce relapse

granulocyte-macrophage mobilized PSC

cells that can inhibit T cell immune function is a balance

tive regulators,

cells

restoration.

immune function PSCT are needed

we found that factor (GM-CSF)

contain Because

of tumor

immunological

remission

Recently stimulating

etic and

Omaha

to inadequate

prod-

trans-

Center,

strategies to enhance mune tolerance after

one traruplantation apoptosis

Medical

also

as pu-

a statistically

Furthermore, in CD14’, enriched of T

charwere

T cell function

E. Talmadge

optimal

colony-stimulating

peripheral peripheral

transplantation

stem cells inhibit

found

manipulation reconstitution

postof stem after

PSC.

INTRODUCTION Myeloablative,

high-dose

therapy

(HDT)

followed

by autol-

ogous peripheral is used for the

blood treatment

stem cell transplantation of advanced malignancies

(PSCT) [1, 2].

Myeloid recovery is more rapid following PSCT compared with autologous bone marrow transplantation (AuBMT) when the peripheral blood stem cells (PSC) are collected after

mobilization

motherapy,

or

with both

[3].

hematopoietic In

growth

addition,

PSCT

factors, results

Patients Between

April

nancies

Abbreviations:

fraction;

retrospective

study

signfficantly de[7]. Further-

demonstrated

that

the

failure-free survival (FF5) of lymphoma patients after PSCT using steady state PSC products was superior to that observed after AuBMT [2]. However, a high relapse rate is still observed after PSCT and only a minority of patients achieve long-term disease-free survival lapse following PSCT may be attributable

October,

1995,

candidates

for

[1, 2]. Disease renot only to sub-

PSC,

HDi

glutinin;

turns to pretransplant pressed compared one

were

21

patients

HDT

with

and

advanced

autologous

malig-

PSCT

were

chein an

transplantation;

more,

and

who

earlier reconstitution of the immune system compared with AuBMT, perhaps due to the large number of lymphocytes in the PSC product [4-7]. Although immune function relevels, it remains with normal individuals

METHODS

failure-free

factor; PBS, PE,

hyde;

TCR,

isothiocyanate;

phycoerythrin; T cell

TNF-a,

tumor

natural

suppressor.

necrosis

Correspondence: ogy

and

South

42nd

Street, September

31,

Journal

receptor; factor

WBC,

saline;

BSA,

Omaha, 11,

phytohemagstem

bovine

terminal

graft

of

1996;

revised

serum

albumin; transinterferon-y;

host

disease;

Department Medical

January

27,

FR,

paraformalde-

IFN-y, versus

Ph.D.,

68198-5660.

cell;

PFA,

Nebraska

NE

blood

deoxynucleotidyl

interleukin-2; GBHD,

cell FF5,

colony-

white

allophycocyanin;

E. Talmadge, University

PHA,

granulocyte-macrophage

IL-2,

a;

cells;

peripheral blood marrow transplantation;

blood; TdT,

APC,

James

Microbiology,

Received January

peripheral

stem

PSCT, bone

GM-CSF, PB,

phosphate-buffered

fluorescein

ferase;

blood

therapy; autologous

survival;

stimulating Flit,

peripheral

high-dose AuBMT,

NS,

of PatholCenter,

1997;

600

accepted

1997.

of Leukocyte

Biology

Volume

61,

May

1997

583

studied.

Twelve

lymphoma. ease,

and

sent

of these

seven one

was

had

obtained infusion

tg/m2

to mobilize

PSC

apheresis 10,000

tration).

PSC

were

In this

study,

obtained

of the

All

patients

the

days with

fresh

to the

cell

approved

granularity)

the

use

were

NC)

centrifugation

were

layered

47.5,

52.1,

at

by

PHA

either

the

second

donors)

Mononuclear

were

Institutional

each

the

61.1,

Review

on

interface;

interface

and

immunomagnetic

cell

separation

cells

(up

(PBS)

to

x

1

in the

108)

identified

was

were

interface;

mm

15

collected

by a magnetic

or

cells.

CD8+

with in

cells

separation

38.6

FR6-7,

use

the

or CD8

Briefly,

monoclonal

used

analysis

Briefly.

with

cell

either

in

100

fix

(40%

PE

tL

or

were

biotin-APC,

of ice-cold solution

in

on

cells

were

permeabilized

trate

solution

were

beads

as purified

resuspended

dry,

in

a

and

purchased

incubated

25

quency

using

a 0.1%

tL

from

96-well

dead

cells,

debris, individually scatter.

The after

cells

plot

was

and

for

60

was

used

aggregated

analyzed

cells.

and

plotted

frequency

of non-apoptotic

h of incubation

in

10.03

TdT

were

the

cells

and

CD8

PHA

stored

cells

+

then

forward

of each

antibodies

erythrin

and

(Coulter,

(APC)

using

isotype

controls

cells.

All data

584

Journal

were

as

or

was

used

acquired

analysis

was

of Leukocyte

stained

or

cytes

(isolated

alone

with 72

with

saturating

cell

anti-CD13 FL)

to determine

performed

Biology

levels

surface

with

the

San

streptavidin

thresholds

for

(Becton-Dickinson)

using

Volume

CELLQUEST

61,

May

harvester allowed

were

to

counted

mitogen

of

stimulus).

cGy)

optimal and

CD8

cells

co-culture

the

in triplicate. to the

in tested

wells

co-cultured

final

The

following

with

5%

x

cells)J

(0.5

tg/mL)

atmosphere.

cells

was

in a

cultured medium

The

mitogenic

determined

by 3Hthy-

All experiments activity

% suppression

cells)/(mean

also

were

CO2

frac-

0.25:1 lympho-

in complete

inhibitory

formula:

co-cultured

ratio)

18 h ofculture.

percent

PSC and

autologous

cultured

or responder over

a 1 :1

were

h at 37#{176}Cin a humidified of the

at

Cells

(each

varying

0.5:1,

of PHA or

lym-

with

cells 1:1,

allogeneic

Ficoll-

autologous

co-cultured

of 2:1,

inhibitory

Briefly,

or isolated

concentration

as a control.

11J.

inhibitory

ratios

The

T cell

10,

were

putative

cell assay

to measure

PBLS

(I:R)

CD4

incorporation

was 1

=

cpm

were calculated

(mean

-

in control

wells

cpm without

100.

Paraformaldehyde mechanism

a 1% fixed

mm

of inhibitory

were

with

activity (PFA)

at room

cells

examined

using

solution

in

1640

After

washing

temperature.

used

instead

responder

Transwell

was

RPM!

of irradiated

PBLs

and

T cell

three

cells

cells

fixed

times,

(w/v)

these

in co-cultures

inhibitory

with

medium

activity

PFA-

(96-well

was

evaluated.

The

with

co-culture

co-culture

mechanism

of T cell

using

a 96-well

(Nunc,

between

from

fled

the

T cell

(10

cubated

for incubated

salted

out

staining

100%

ethanol.

positive

titated. by

y

for the

was

extracted

washed,

of 10 gel light,

tg

and

and

cell-to-cell

cells.

were 0.1

photographed.

analyzed

et al.

f23J.

pelleted,

50

addition

of DNA

containing

0.2-tm

to prevent

tg/mL

then

5

per

mm with

lane

a modi-

Tris-HC1

tg/mL

M) and

washed pig/mL

mM

of proteinase

of 50

(0.5

using Briefly,

after

ap-

in

lysis

pH

7.5,

resuspended

50

and

of NaCI

samples

incorporating

responder

Mollereau

1 h at 37#{176}Cand addition

DNA

Samples agarose

ultraviolet

suifate,

1 h at 50#{176}C.After

were

and

ethylenediaminetetraacetate, dodecyl

examined

system

by DNA fragmentation

by were

also Denmark)

cells

cells

cells

mM

allo-

software

inhibitory

co-cultured

treatment sodium

1.2%

Roskilde,

as described

0.57%

was chamber

of apoptosis

procedure

propriate

inhibition

transwell

membrane

anti-

and

1997

treatment

paraformaldehyde

for 30

buffer

phyco-

Background

Plus

of

markers: MA),

(Becton-Dickinson), added

Life were

incorporation

(no

7,

cells

microplate.

PHA

midine

DNA with

(Becton-Dickinson,

fluorochrome.

on a FACStar

blocking

Cambridge,

samples

h For

cells

were

Specific

assay

responder

(500 of an

response

contact

immunopheno-

After

The

of a 96-well filters

cells

described

(allogeneic)

as

ofirradiated

Anopore

CD8

calculated.

11J.

following

anti-CD8 third

surface

flO,

the

Hialeah,

the

for cell

Diagnostics,

biotin-conjugated

or anti-CD19

data

for

(T-cell anti-CD4

phycocyanin

detailed

used product were

specffic

(PE)-labeled CA),

was PSC

of cells

anti-a3TCR

FITC-labeled

CD14,

of the

aliquots

monoclonal

Jose,

analysis

fraction

immunoglobulin,

10)

Measurement

cytometric

typing

x

flat-bottom

studies

Flow cytometry Three-color

h).

T cell inhibition

co-culture

normal

presence

plates)

scat-

were

CD4 then

54

fre-

excluding

versus

was

reand

The

A forward

apoptotic

or

then

while

the

previously

at inhibitor-to-responder

The

mM all

analysis.

expression

or

media

imol/ 25 IN)J

as follows: all

CD4 as

tubes

cicells

(TdT),

cytometric

to gate

back-gated 24

The

flow

for been

(1

according and

the

(Indianapolis,

mm.

fix before

PHA.

(Amersham

the

control

i0

The

2 p1

transferase

for

tg/mL

96

use

and

x

Becton-

cultured

0.5

The

counter.

with

and autologous

performed

sodium

mixture

the IL).

added,

beta

compared

or

(total

with

Grove, was

multi-well was

Hypaque-purified

in the

added

washing,

imol/dATP,

Mannheim

bath

apoptotic

scatter

3

was

Packard

has

anti-

of FACS

X-100/0.1%

reaction

gran-

at 1

(Falcon,

were

2.5,

added

filters

cocktail

of PHA plates

Cells

5,

was

Downers

methodology

were

resuspended

temperature.

After

deoxynucleotidyl Boehringer

at 4#{176}C in FACS

side

Triton 2 mm.

of TUNEL

and

at room

or

of CD14).

(3Hthymidine/well

IL)

fiberglass

scintillation

numbers

labeled

microliters

formalin)

mm

30

(FITC)-dUTP,

U terminal

ofspecific

by

buffered for

in a 37#{176}Cwater dark

10%

at 0#{176}Cfor

in 50

agents

cells

of

hundred

side from

expression)

presence

NJ). of

1 .tCi

Heights,

onto

Allogeneic

tion)

CD4+

antibodies

in PBS.

One

shaker

isothiocyanate

CaCI2,

ter

PBS

a horizontal

specific

twice

BSA.

by incubation

fluorescein

with

washed

PBS/i%

incubated

in the

stained

CD14

microtiter

absence

culture

Instruments,

air

with

by flow cytometry

suspensions

with

distinguished

expression

in the Park,

or

h of

harvested

for

TUNEL

18

phocytes

saline incubated

to magnetic

and

scatter

(intermediate

clearly

negative

lower

cultured

Lincoln

Arlington

activity

Mini-

phosphate-buffered

bound

column

of the CA).

and

and

and

flat-bottom

presence

3HJthymidine

PSCs,

Auburn, (BSA)

cells

were

side

markers

1

the

and

the

Inc.,

anti-CD4

at 6-12#{176}C. The

fraction

FR2,

were

96-well

the

final

The

albumin

microbeads-conjugated

for

cells

Percoll-fractionated

Biotec

serum

at 38.6,

layer. from

scatter

scatter

Labware, in

(Packard

PSCs

(FR)

follows:

to 61.1%

resuspended

bovine

as

performed

(Miltenyi

population.

centrifugation,

isolation;

70.1%

Durham,

Ifractions After

Percoll

T cells

sorting

0.5%

magnetic

body

47.5

CD8

system

containing

the

pellet

CD4

gradient

38.6%

the

PSC

70.1%J. and

of the

FR3-5

to the

To purify

Percoll and

collected

top

in

Dickinson either

Teknika,

as an unfractionated

65.6,

were

band

to 47.5%

MACS

used

on a discontinuous 56.5,

interface

(FRI). 61.1

and

cells

cells/well

Center.

(Organon

monocytic

of CD14)

using

specific

mitogenesis

Sciences,

Ficoll-Hypaque

side

gated

for the

count adminis-

and separation

separated

CD14

side

(higher

was

intensity

expression

(lower

ulocytes

subpopulation

fluorescence

technique;

high

lymphocytes

then PSCs

of this and

the

Cell isolation

and

Lakewood,

normal

by the

Medical

(WBC)

BCT,

from and

of

blood.

Each

(cell scatter

a dose

at

of GM-CSF

products

of Nebraska

a continuous

(Cobe

patients

con-

peripheral

blood

initiation

dis-

informed WA)

the

spectra

PSC (both

protocols

University

white

after

a Cobe

All samples

into

(Becton-Dickinson).

Hodgkin’s

received

Seattle, cells

when

non-Hodgkin’s

had

Written

(Immunex,

(3-4

we used

one

leukemia.

stem/progenitor

collected

grade

cancer,

patient.

started

according

Board

each

cells4tL

apheresis.

intermediate

breast

of GM-CSF

was

reached

had

risk

myelogenous

from

intravenous

or third

high

acute

250

CO).

patients

had

were

ethidium

RNase

K), and

at 70#{176}C.Proteins DNA 70%

precipitated ethanol

electrophoresed bromide,

in-

A, samples were with

and

quanon

visualized

a

Semi-quantitative analysis of cytokine [reverse transcriptase-polymerase chain reaction (RT-PCR)]

mRNA

quences

were

analysis.

The

ments

N Total

cellular

state

or

reagent

RNA

(24

Gaithersburg,

procedures. at 260

precipitated stored

at

of

(GOBCO-BRL), 1 p1 l1T at

(10

of lOx of dTTP,

(GIBCO-BRL) Each

tration

of 250

sterile

dH2O.

DNA

thermal

annealing

cycler

genes)

for

other

genes.

PCR

buffer

and

visualized Rochester,

(Genosys, liquid

within

the

pairs

in length,

intron

so that

be

readily

had any identified.

purified

region similar

such

amplification

cycles The

they

blot

on

were

(-

oligonucleotide

probes

20

TABLE

1.

The

the

Relative of cells

samples

gene

with

to be corn-

signals

sample/experiment. cytokine

strin-

(Phosphor-

number

housekeeping

of each

between

Cytokine

signal

to the

sig-

signal

from

-actin.

t-test

than

0.05

was

correlation

SPSS 7.0

using

of less

base

was

used

to compare

coefficients

for Windows taken

were

(SPSS,

control

obtained

Inc.,

and

by the

Chicago,

IL).

experiPearson’s

A P value

as significant.

of monocyte was determined

subpopulation

was

intensity

and

gated

for CD14,

lymphocyte by fluorescence using

CD4,

side CD8,

se-

cytes

Oligonucleotide

were

Primers

in the

and

higher-density

fractions

phenotypes cytometry.

scatter CD19,

receptor (TCR) a/ expression. Monocytes were found in the low-density fraction (FR2),

would

exon

frequency products

rescence

a single

DNA for

Student’s

groups.

Each

chosen

flanked

genomic

unpaired

mental

The PSC

published

strand)

approximately and

of contaminating

the

ratio

probing

digital

analy-

commercially

based

autoradiography.

an equal

translation) under

RESU LTS

high-performance

3’ primers

gene

CA)

cit-

were

specificity

for Southern

as the

housekeeping

if the

in a given

nick

by

and

sodium (Stratagene).

washed

analyzed

by using

blotting,

h,

ammem-

the

(Kodak,

synthesized

expressed

saline

(by

24

0.25

The

to nylon

x

frag-

with

N NaOH.

oven

32P-labeled

Sunnyvale,

used

in 2

for

the

denatured 0.4

transferred

washed

and

obtained

not

were with

blot

blotting

in TAE

gels

the

undetectable

method all

for

gel

ig/mL).

temperatures,

Specific

40

to confirm

were

and that

melting

different

trans-illuminators

designed

strand)

at

were

were

The a

55#{176}Cfor

agarose

by reverse-phase

were

(+

and

with

using

set and

cytokine,

Dynamics,

Results

in Southern

by

Statistics

concentL

with probes

amplification,

are

were

use

h in a hybridization

internal

were

pared.

the

(GIBCOto 50

CA)

2%

processed 1)

mix mM

1.5

Molecular transcripts

gels

passively

hybridized each

groups nals

of

primer

IFN-a

and

were

of dNTP iiL

for

simultaneous

the

amplification

(0.25

and

cDNA

Imager. mRNA

of super-

polymerase

3-actin

1 h

conditions

were

for 4-18 were

for verified

neutralized

membranes

oligonucleotide

gent

HT buffer

1 tL

a final

on

and

dGTP,

strand

City,

ultraviolet

were

Primers

5’ primers coding

for

x

specific

and

sequences

transfer,

was

Briefly,

dH2O,

prehybridized

fashion

product

membranes.

The membranes

re-

by incubating

adjusted

and

studies

and

chromatography. with

Foster

(Table

5

1.5

to DNA

bromide

gels

TX)

was

separated

quantitative

42#{176}Cfor

of

1 tL

volume

!L-2

using

at

After and

in a similar of the

with

denatured

brane. rate

by aband

dCTP,

first

DNA

rec-

nylon

washed

plified,

of Trizol#{174}

ethanol

and

to give

steady

100%

stopped

tL)

cycles were

primers

Houston,

sequences

of Taq

ethidium

sequences,

Oligonucleotide

vol

dGTP),

subjected

of 20

with For

amplified

tL

Elmer,

photographed

NY).

and

(60#{176}C for fragments

aliquoted

(GIBCO-BRL),

total

(Perkin

determined

dATP,

of

dCTP, (2.5

was

a total

stained and

buffer 0.25

temperatures

was

microliters

added

mixture

was

microliter

each

use

were

synthesized

mM

and

manufacturer’s

(GIBCO-BRL),

Two

The

by

the

2.5

One

reaction

dATP,

pmol/mL.

other

(10

PCR

was

The

mix primer

and

primer

dH2O.

The

mm.

and was

in

PSC

products per

of RNA

DNA

(dT)18

each

BRL).

from

dNTP

from

concentration acetate

tg)

(GIBCO-BRL).

MgC12

sis.

1 tL

to 5 iiL mM

of the

(2

10

PBL

micrograms

RNA

70#{176}Cfor

added

RNA

Two

isolated

MD)

3 M sodium

oligo

was

normal

70#{176}C. First-strand

-

tL

dTTP),

h)

The

nm.

in 15%

4.5

mix

cells

(GIBCO-BRL,

sorbance

script

106

PHA-activated

ommended

using

from

onto

HC1,

synthesized specificity

and and

in

fluoT cell

(CD14 cells) while lympho-

(FR3-5).

The

re-

Probes

Cvtokine Primers

IL-2

CAA GCA

CTC TCC

CTG TGG

TCT TGA

TGC GTT

ATT TGG

GC G

IL-4

ATG GTC

TGC TGT

CCG TAC

GGA GGT

ACT CAA

TTG CTC

TC G CG

IFN-y

CAG

GTC

ATT

CAG

ATG

TAG

GCT

TTT

CGA

AGT

CAT

CTC

C

3-actin

TGA

ACT

GTG

ACG

TGG

ACA

TC

TNF-a

ACT GAG

CGT CTG

CAT AGA

ACT GAT

CCT AAC

GCT CAG

TG CTG

GTG

CAG

ATA

CAT

GGG

CTC

ATA

CCA

GGG

IL-b

ATG TCT

CCC CAA

CAA GGG

GCT GCT

GAG GGG

AAC TCA

CAA GCT

GAC ATC

CCA CCA

IL-2

GCA

CTT

GTC

ACA

AAC

ACT

IL-4

GCG

ATA

TCA

CCT

TAC

AGG

AG

IFN-y

GCA

TCC

AAA

AGA

GTG

TGG

AG

TG

AAG TTT

CTC GAC

mo

et al.

Probes

CC

3-actin

CAA

CAT

CAT

TGC

TCC

ICC

TNF-a

CCC

TCC

ACC

CAT

GTG

CTC

CTC

IL-b

CAG ATC

GTG TAC

AAG AAA

AAT GCC

CCC ATG

TTT ACT

AAT GAC

CAA ATC

CD14

CAG

cells

AAA

in

CCC

mobilized

PSC

products

585

TABLE

Cell

population

CD14

2.

Cellular

monocytes

CD4

T cells

CD8

T cells

22.3

±

2.0

19.3

± 2.1

6.6

±

0.49

± 0.24

±

1.9

6.3

±

1.8

4.3

± 0.9

NDb

0.18

± 0.12

± 4.3

10.5

± 4.1

ND

0.83

± 0.4

0.5

± 0.1

ND

ND

0.7

0.5

± 0.1

ND

ND

Unfractionated

41.1

± 2.7a

FR2

71.1

± 3.4

FR3-5 Purified

CD4

Purified FR2

CD8 after

depletion

CD4

and

FR3-5

34.5

PSC

Fraction

Granulocytes

CD19 1.8

4.1

± 1.1

38.3

± 3.2

0.7

± 0.2

92.7

±

0.7

± 0.4

8.6

± 1.7

±

1.7

0.6

± 0.2

1.9

± 0.5

5.9

± 2.1

±

1.1

3.6

±

9.6

± 3.5

18.7

± 6.6

1.1

2.4 88.8

±

ofCD4

7.0

± 2.1

86.8

1.2

0.31

± 0.20

and

CD8

5.1

1.2

20.8

± 3.1

1.77

± 0.80

SE.

not determined.

found

using

autologous

CD4

and

CD8

cells

phenotyping in each fraction are sumIn the unfractionated PSC products, of CD14, CD4, and CD8 T cells 19%, respectively. After Percoll separa-

tion and addition to co-culture cells. These studies used an

with highly admixture

(MiniMacs)

and

tion,

were

PSc

CD14

lation

.

enriched

to 71%

CD84 cells from FR3-5, showed high enrichment

(93

and

CD4 and was highly maining

2

cells

CD4 and separation,

89%,

CD8 enriched

FR3-5

CD8

few

in a relative

granulocytes,

of

population and the re-

CD14,

CD4,

enrichment

and

of B

a/

CD4CD8

PSC

mal PB 0.001).

mononuclear After Percoll

cells (101,256 separation,

FR3-5

(which

CD4

had

had

± 6,486

a 4.1

a depressed PHA cpm) compared

of T cell

response

that of normal ogous T cells which

± 7,214 cpm; lymphocyte-enriched

1.7%

±

(0.5 ig/ with nor-

CD8

was

to PHA

was

aliogeneic cells had a statistically

monocyte

contamina-

cells

from

the

inhibited

>80%

observed

role

cells for

by the

from PSC products T cell proliferation.

of CD14

cells

in the

compared

with

(Fig. 1). The isolated autolnormal response to PHA, isolated

a 2:1 I:R ratio (n 7). In all studies cell proliferation was dependent on cells in the co-culture. Thus, these CD14 hibition

P

isola-

CD14

cells

at

the inhibition of T the ratio of CD14 results indicate that

are the Further

inhibition

source support

of T cell

of the infor the function

is

had an increased PHA response (50,050 ± 8,741 which was still lower than that observed with normal

PBL (P purified (78,564

0.001). However, CD4 (87,602 ± ±

unfractionated ence in the with normal cells, T cell Unfractionated activity dependent

9,215

cpm)

PSC (P mitogenic

was

the PHA response 20,120 cpm) and increased

0.001) response

and was

in both the CD8 cells

compared

with

a further enrichment for which showed a significant

no significant differobserved compared

FR3-5

Purified

ci+

1’

Purified

CD8+

#1

CD4,8-neg.

FR2

cell inhibitory activity (94%) compared cells (P 0.001). In contrast, the FR3-5 and purified CD4 or CD8

Normal

with unfractionated lymphocyte-enriched T cells had no inhib-

-

-40

1.

itory

cells

I+

.tg/mL

Biology

Volume

61,

May

1997

-20

0

I

I

I

I

20

40

60

80

I

100

% T cell Inhibition

Fig.

activity. Furthermore, the B and CD4CD8TCRa/ cell-enriched populations in the CD4and CD8depleted FR3-5 cells also did not inhibit allogeneic T cell proliferation. Similar results (Fig. 2) regarding susceptibility to CD14 cell inhibition of T cell blastogenesis were

of Leukocyte

PBL

in T

itory

Journal

I#{174}

CD4,8-neg.FR3-5

inhibitory activity (87%) PSC. After T cell depleCD14 cells was found increase in allogeneic

I.

FR2

PBL, suggesting that in the absence of CD14 functionality in these populations was normal. PSC cells had significant T cell inhibitory

FR2 had increased with unfractionated

I#{174}

Unfractionated

the

(71%) at an I:R ratio of 2:1 (Fig. 1), which was on the I:R ratio (results not shown). The CD14

cell-enriched when compared

586

autologous

after

purified CD14 (1:1) of isolated

products and >99% pure CD14 cells. The highly purified monocytes were obtained by Percoll density gradient centrifugation and cell sorting by flow cytometry. This resulted in a significant and cell ratio-dependent autologous inhibition of T cell proliferation. A similar inhibition

=

The unfractionated mL) response (31,365

tion FR2,

Purified

depletion

FR2 (87%),

contained

resulting

cells),

After

the remaining CD14 cells

population

in FR2.

by immunomagnetic of each T cell popu-

respectively).

cells, for

T cells,

cells (CD19 T cells.

tion) cpm),

± 0.36

± 0.3

sults of the cellular marized in Table the mean frequency were 41, 22, and

or

a/I3TCR CD4CD8

B cells

depletion

#{176}Mean± 6ND:

in each

of

CD8

after

7.2

Phenotyping

Normal

tionated

fractions)

of PHA

3Hthymidine Each bar normal

responder

(PSC

for

4 days.

incorporation represents mean PBL PSC

(P < 0.01). (P

0.001).

PBLS at

an

were I:R

The

of

mitogenic and

±

co-cultured ratio

SEM.

Significant

the

2:1

in

with

irradiated

inhib-

the

presence

of

response percent

Significant

decrease

was inhibition

increase

compared

0.5

determined

by

calculated. compared

with

with

unfrac-

the significant quency

and

0.936,

P

(Fig.

inhibition

0.001). for cell-cell

role

A

correlation the

3) between

of allogeneic

CD14 T cell

cell

fre-

activity

(r

100

=

>

co

contact,

compared

with

soluble

= U) 0)1!) 0! 0.

fac-

in the inhibition of T cell activity was suggested by experiments using culture supernatants and transwell studies that were negative for inhibitory activity compared with partoys,

allel co-culture assays (Table 3). The cell contact was directly demonstrated autologous CD14 and isolated CD4 PFA-fixed

low

density

purified CD4 cells, cell activity (75.1% 0.5:1).

In contrast,

PSC

cells

showed at I:R

=

when

the

0.

requirement for cellusing PFA-treated and CD8 T cells.

(FR2),

but

significant 2.1 and

cells

U)

from

not

PFA-fixed

T cell 14.2%

inhibitory at I:R

PSC

products

Cl)

-50

0

20

40

60

% CD14

80

100

cells

=

Fig.

were

the

3.

Correlation

frequency

between

of CD14

inhibitory

cells

within

cell the

function

Percoll

(I:R

2:1)

=

separated

and

fractions.

co-cultured with normal PBMC in transwell chambers, T cell inhibitory activity was completely abrogated (- 7.0% at I:R = 2:1 and -9.3% at I:R = 0.5:1; Table 3). Studies of T cell hypodiploidy (results not shown), DNA fragmentation (Fig. 4), and TUNEL analysis (Fig. 5) suggest that apoptosis role of

is involved in the mechanism of cytotoxicity. apoptosis is suggested by the studies using

cultures of which results trast,

CD14 cells and T in DNA fragmentation

CD14

tion

did

cells

not

Similarly,

and

result

lymphocytes

co-cultured tion (lanes

without

PHA

fragmentation

alone,

(Fig.

monocytes

stimula-

4, lane

alone,

cells

and

41%

of CD8

5).

(24

h) PBL

of

interleukin-2

TNF-a,

IL-b,

PSC

and

products the

absence

CD8

cells

a possible

4).

of PHA

were

both

IFN-’y

TH-2

mRNA

the

6.8%.

transcripts normal

with

Furtherexpression

PHA-stimulated

PBLS.

higher PBLS,

in the PSC suggesting

Overall,

products PBL than

observed

PBLs. higher

was also significantly with PHA-stimulated PSC

(IFN-’y),

were

significantly

compared

increased

interferon-y

with

had

phenotype.

mRNA from the that of PHA-activated

Significantly

(IL-2),

IL-4

product

The level of IL-4 products compared

and

(Table

compared

PSC

of IL-b

100

in the

and

expression

more,

became

CD4

stimulated

that, after of PHA,

cells

In contrast,

of apoptotic

To determine the expression of potential immunoregulatory cytokines, we analyzed the expression of cytokine genes in the PSC products compared with normal or PHA-

in

PHA

(Fig.

percent

fragmentaof CD4

and TUNEL analysis revealed of PSC products in the presence

of CD4

4).

and

lymphocytes did not result in DNA 1, 2, and 3, respectively). Backgating

or CD8 cells the co-incubation 41.2%

lymphocytes with PHA, (Fig. 4, lane 5). In con-

lymphocytes

in DNA

The co-

apoptotic

the

levels

of cytokine

more closely that of steady

resembled state PBL

cells. *

80

DISCUSSION :

*

60

The inhibition of irradiated

-C C .

U

*

and

20 [18],

[20],

tion,

0 1:1

2:1

0.5:1

spleen cells from mice after total lymphoid irradiation undergoing chronic graft-versus-host disease (GVHD) or after cyclophosphamide treatment [21]. In addiYoung

et al. have

poiesis-associated by GM-CSF

l:Rratio

and

reported

2.

T cell

CD8

cells

fled

to >99%

onescent

was

inhibitory

activity

examined

with

purity

activited

>90%

purity

isolation. co-cultured

by Percoll cell

by

The

sorting.

Percoll

CD4 with

for highly

the

gradient and

CD8

CD4

cells monocytes

admixed for

72

pun-

followed were

followed

were

and

monocytes

cells

centrifugation

CD8

purified

autologous CD14

centrifugation

CD4

gradient and

purified purified

by

by

to

MiniMACS

at a 1:1 h with

flu-

isolated

0.5

ratio tg

and PHA

cell inhibitory activity uct is not unanticipated.

ported cell

that non-mobilized mitogenesis [11]

the

mitogenic

poration. crease cytes

Each compared (P

0.01).

response bar with

was

represents CD4

determined mean

and

CD8

±

cells

by SD

(n

without

3Hthymidine =

7). the

‘K

Significant

addition

inconde-

of mono-

prior

to mobilization

inhibitory associated

lao

CD14

of myelo-

cells is stimulated the observation

in GM-CSF-mobilized We recently reported

nor [10].

activity in PSC with mobilization

et a!.

the induction

suppressor 15]. Thus,

tory activity for T cell function mobilized PSC products from the PB following autologous

T

and

that

immune IL-3 [14,

T

Fig.

as the ability of allogeneic or

autologous lymphocytes to PHA mitogenesis. This activity is found with human [12, 19] and rodent BM cells [13, 16],

40

I-.

of T cell function is measured cells to depress the response

cells

is present in both GM-CSFcancer patients [11] and in PSCT [7, 10]. We also re-

PSC did

had cells

We

suggest,

products and

in

of

PSC prodthat inhibi-

mobilized

little from

ability to inhibit the patient’s PB therefore,

(or their leukapheresis

PSC

that

frequency) and

producta

the is may

587

TABLE

3.

Role

of Cell-to-Cell

Contact

for

T Cell

Inhibitory

Cell

Activity

% Inhibition’ Inhibitory

cells

PSC

Normal

PSC

Transwell

Low-density

PSC

Low-density

No

PSC

Purified

CD4

cells

b Non-irradiated Values

dient

inhibitory

are

1l:R.

mean

inhibitor

centrifugation

tion. The proliferative

culture’

PFA

consistent

our

T cell

9.3

±

1.8e

91.6

± 6.0

25.0

± 9.1

±

14.2

treatmentb

75.1

PFA

treatment

-1.5

with

were

irradiated

fixed

with

±

from three ratio.

SEM

1%

independent

decrease

compared

with

normal

decrease

compared

with

no

observed

inhibitory PFA

cells

for 30

mm

4.4

± 8.1 for 72

and

h in culture

similar

T cell

0.5:1

±

2.6

-8.7

±

2.8

plates

with

inhibitory

cell

(or

assay

report

[11J.

In

and autologous products would

function

treatment

pe-

3

gra-

cell

isola-

decreased normal PBL addition,

the

PHA mitogenesis suggest that post-

is depressed

2

(P (P

coculture

PFA

in the

bead

had a significantly compared with

previous

experiments. 0.05). 0.05).

by the

infused

CD14

cells.

However,

the

immune

re-

sponse

immunomagnetic

PSC to PHA

I

2.6e

PFA

Significant

of allogeneic from the PSC

transplant

±

With

1Significant

and

with

inhibition by cells

6.9

-7.0

treatment

phenotype of the apoptosis-inducing PSC products by Percoll density

unfractionated response

±

With

cells

to responder

to the immunosuppression blood post-transplantation.

To determine the cells, we fractionated

25.7

82.7

performed.

(

contribute ripheral

I:Rd

4.2

I:Rd

coculture

(1 Responder PBMCs were cocultured without) inserting transwell chamber.

was

2.1 ±

Treatment

(at

4

least

to PHA of purified CD4 or CD8 cells from the PSc products was normal, suggesting that unstimulated T cells remain functional. Furthermore, the CD14 cell frequency correlated directly with the inhibition of T cell activity. These type of cells

results demonstrate with T cell inhibitory

products

a CD14

is

cell

that the primary phenoactivity in mobilized PSC

that

may

monocyte or a monocytic-dendritic This is consistent with our previous mobilized

in part)

PSC

cells

from

healthy

be

either

a mature

cell precursor observation that donors

with

a low

[24]. nonfre-

quency of CD14 cells (10%) had significantly lower levels of inhibitory activity compared with GM-CSF-mobilized

5

PSc

[11].

The cellular cells has been

origin of cells with inhibitory controversial, with few studies

activity for T that have fo-

cused on human cells. Strober et al. demonstrated that one phenotype with this activity in both human and murine BM is a CD4CD8c43TCR T cell, which they termed a natural

suppressor

(NS)

ously characterized ular lymphocyte

cell

[12,

16].

Other

NS cells as a member family [17], null cells

monocyte lineage [13], or hematopoietic [14]. However, in the present study, depleted cells from FR3-5 that are

CD8TCRa/I sor cell

by carbonyl-iron activity ( - 26

PSCs (61 ± cell function

T

Fig.

4.

cells

were

washed,

CD14

cells

induce

coincubated and

alone;

lane

2, CD14

cytes;

lane

4,

co-culture;

with

analyzed

lane

for

activation-induced lymphocytes DNA

and

with

or without

fragmentation.

monocytes

monocytes

T cell

alone;

Lane

lane

lymphocytes

5,

monocytes

and

of

Leukocyte

Biology

apoptosis. PHA 1,

CD14 for

3, PHA-activated without

lymphocytes

PHA

with

Journal

Volume

61,

h,

PHA

after

May

1997

24 24

h h

10%

±

at a 2:1 (59,905

progenitor cells CD4and CD8enriched in CD4

immunosuppression studies from our of phagocytic cells

phagocytosis abrogates 8%) compared with ±

I:R ratio) 14,073

vari-

suppresthe role of cancer laboratory from PSC T cell inuntreated

and partially restores cpm) compared with

untreated PSCs (26,905 ± 5,128 cpm) in a PHA mitogenic assay. This observation supports the suggestion that CD14 monocytes are associated with the loss of T cell function in human PSC products. We believe that this

lymphoafter

co-culture.

588

24

lymphocytes

have

of the large gran[22], macrophage/

cells (1.8% on average) had no Several reports have demonstrated

activity.

of mature monocytes in the patients [25, 26]. Unpublished have shown that the depletion products hibitor

studies

CD14

cell

based on Several

activity the cellular investigators

differs

from

conventional

NS cells

[12]

phenotype and phagocytic nature. have reported that suppressor cells

no PHA

cells

CD4+

cells

CD4+

PHA

#{149}.‘i#{149}_ 6.8#{176}h

P. IL

U.

8(SI

A

ii

10

;1

100

1o2

;2

CD8+

cells

i?

AB

AB

no PHA

CD8+

cells

PHA

§ §

F

h. 0.

100

;i Ap0HB

Fig. on

5.

PSC

CD4

or

products CD8

were

and

the

in rodents or rabbits hibit T cell functions that sion

with

of

nitric

macrophage-derived been suggested been

[33, apoptosis studies,

oxide

quirement

and T

[29-31];

of T cell no role

or without

PHA

cells

membrane-associated

required

for cell-cell

contact

that

was

contact. and

in T cell

sis is reported. Speculatively, a lack ofco-stimulation regulation of fas ligand rather than soluble factors be involved

in the

induction

of T cell

h and

stained

with

CD4.

CD8.

apoptosis

cells

in human

using

HTPCR

and

in also has

et al.

PBLS. However, of T cell function

products.

TUNEL.

The

cells

a re-

apoptoor upcould

Preliminary higher

and

IL-b activity)

were

to

reduce

transplantation. ported

that

of T cell the reason planted

mo

the

backgated

BMT inhibitory for the

et

could [22,

al.

35]

with the inhibition to result in a sol-

also

and and

have

allogeneic

et a!.

CD14

PSC

cells

clinical

with

Palathumpat GVHD we suggest

products

in mobilized

et that

po-

allogeneic

in murine

cell activity in PSC decreased GVHD seen

using

2)

expres-

PSC products with normal

associated

prevent

(Table

of mRNA

in human compared

function

GVHD

Sykes NS cells

of allogeneic

studies

levels

this does not correlate and would be expected

uble inhibitory activity. Cells that depress T cell tential

by CD14

PSC

demonstrated

sion for TNF-a, IL-4, (with apoptosis-inducing

In our

observed

results

in-

TNF

[32-34]. Wu monocyte-induced

cell-cell

factor

for 24

the suppresThe involve-

cell inhibitory activity has although recently apoptosis

for a soluble

.tg4tL)

factors that prostaglandins

can mediate patients [25].

as a mechanism that human lines

(0.5

determined.

can produce soluble [27, 28]. For example,

proposed demonstrated

34]

ApoHB

of apoptotic

are released by monocytes of T cell function in cancer

ment

also

co-cultured frequency

..

I #{243}’

100

1o3

al.

re-

models the

levels

products may be in patients trans[36,

PSC

37].

products

However,

589

TABLE

4.

Transcriptional

Expression

of Cytokines

PSC

n CD14’

35.36

PHA

0.5

tg/mLb

3.93-i

2.30

±

0.74

7.1k

10.4

±

15.6

18,975

±

8,440k 0.161k

0.016

±

0.006

1.135

±

E.l.

IL-2’

0.068

±

0.040

0.001

± 0.001

0.698

±

0.215

E.I.

!L-10’

1.302

±

0.493

0.034

±

0.016

5.983

±

4#{149}4#{216}3e

El.

wNy&d

±

0.143k 0.035k

#{216}#{216}#{216}4 ± 0.002 0.002 ± 0.001

0.571 0.005

±

IL4b,

0.509 0.146

±

E.I.

0.16M 0.001

apercent

CD14+

bAverage

value

cells ±

cell

found

activity

of each

by fluorescence

inhibitory

(I:R

cytokine

activities

cells

are

from

autologous

lized PSCT of superior pared with to the

PSCT.

cytometry.

to the

signal

from

in the

disadvantageous

autologous

in the

frequency

gene

range

to dethe re-

5.

using

E., Kandel,

recovery

after

Marrow

Tmnsplanz.

Roberts,

M.

strategy failure

6.

of mobi7.

cells.

9.

research

ENTS

was

Grant Disease

supported

in part

We thank the individuals processing of the stem cell Drs. Howard Gendelman, Sharp, and and helpful

Rita Young suggestions.

for

by National

10.

Institutes

and Program

Nebraska Grant

involved products. Michael

in the harvesting and Our thanks also to Hoffingsworth, John

their

review

Cancer 97-71.

of the

and

manuscript

1 1.

14.

REFERENCES Kessinger,

A., Bierman,

cyclophosphamide, ripheral stem 2.

3.

ease.

Blood

Vose,

J. M..

590

and etoposide for patients

J. 0. (1991)

followed by with relapsed

High-dose

autologous Hodgkin’s

pedis-

Anderson,

peripheral

J. R.,

blood

stem

plants and allogeneic 9, 277-284. Henon, P. R.. Liang,

bone

Journal

15.

Kessinger,

A.,

Bierman,

P. J., Coccia,

P.

Reed,

H.,

of Leukocyte

cell

transplants,

marrow

autologous

transplants.

Beck-Wirth.

Biology

C.,

bone

Bone Eisenmann,

Volume

61,

marrow

Marrow J.,

May

trans-

Traruplant.

Lepers,

1997

M.,

16.

17.

18.

Gillis,

D..

reconstitution

munologic

reconstitution

stem

blood

immune Bone

Mundy.

J.. Rawling,

following

bone

marrow

peripheral

C..

Ng,

blood

transplantation

and

K.. Juttner.

stem

cell trans-

allogeneic

bone

following

cells

transplantation

as compared

to bone

with

marrow.

mobilized

Bone

peniph-

Marrow

Tmns-

in press.

Klingemann,

graft-versus-leukemia 4, 261-267. Verma. U. N.. Areman, E., Dickerson, S. A., Kotula. P. L., Mazumder, A. (1995) Intenleukin-2 activation of chemotherapy stem

H. C. (1995)

cell

Introducing

transplantation.

peripheral

into

autolo-

.L Henzatother.

blood

stem

cells

for generation

Sacher, R., and growth

ofcytotoxic

effec-

tors. Bone Marrow Tran.splaru. 15. 199-206. Talmadge. J. E., Reed, E. C., Kessinger. A., Kuszynski, C. A., Perry. C. A.. Gordy, C. L., Mills, K. C., Thomas, M. L., Pirruccello, S. J., Letheby. B. A.. Arneson, M. A., Jackson, J. D. (1996) Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation. Bone Marrow Tran.splani. 17, 101-109. Mills. K. C., Gross, 1’. C., Varney, M. L.. Heimann. D. C.. Reed, E. C.. Kessinger, A., Talmadge, J. E. (1996) Immunologic phenotype and function bone

marrow, I.

blood

18,

Transplant.

Schmitz-Wolf,

C.,

stem

cells

and

umbilical

cord

blood.

Bone

53-61.

Dejbakhsh-Jones,

S.,

Ginzton,

N.,

Greenberg,

P.,

Strober, S. (1992) T-cell subsets and suppressor cells in human bone marrow. Blood 80, 3242-3250. Noga, S. J., Wagner, J. E.. Horwitz. R. L.. Donnenberg, A. D.. Santos, C. W.. Hess, A. D. (1988) Characterization of natural suppressor cell populations in adult rat bone marrow. J. Leukoc. Biol. 43, 279-287. Schmidt-Pak, A.. Wright, M. A., Matthews, J. R, Collins, S. L., Petruzzelli, C., Young, M. R. I. (1995) Mechanisms of immune suppression in patients with head and neck cancer: Presence of immune suppressive CD34+ cells in

2322-2325.

E. C., Gordon, B., Armitage. J. 0. (1993) High-dose chemotherapy and autologous hematopoietic stem-cell transplantation for aggressive nonHodgkin’s lymphoma. J. Clin. Oncol. 11, 1846-1851. To, L. B.. Roberts, M., Haylock, D. (1992) Comparison of haematological recovery times and supportive care requirements of autologous recovery phase

4.

cell 77,

P. J., Vose, J. M.. Armitage,

carmustine, transplantation

Comparison of hematopoietic and marrow or blood stem cell transplants.

bone

To, L. B,

eral

Marrow 12.

C. (1992)

autologous

in human

13.

1.

b

9, 285-291.

M.,

factor-mobilized

RO1-CA61593 Research

analyzed

marrow transplantation. Bone Marrow Transplant. 12. 469-475. Kiesel, S., Pezzutto, A., Korbling, M., Haas, R., Schulz, R., Hunstein, W.. Dorken, B. (1989) Autologous peripheral blood stem cell transplantation: Analysis of autografted cells and lymphocyte recovery. Tmnsplant. Proc. 21, 3084-3088. Talmadge. J. E.. Reed, E., mo, K.. Kessinger, A.. Kuszynski. C.. Heimann. D.. Varney, M., Jackson. J., Vose. J. M., Bierman. P. J. (1996) Rapid im-

gous

of Health Smoking

were

software.

autologous

Immune

plantation,

8.

This

Units

quart

Wunder,

plant.

ACKNOWLEDGM

3 actin.

image

C. (1993)

products

inability

of CD14

housekeeping

linear

in

ability that PSC

the

the

PBL.

[38] to replicate the retrospective observation failure-free survival of steady state PSCT comBMT reported by Vose et al. [2] may be due

difference

±

4:1).

=

signal

on their therefore,

Indeed,

O.272

SE.

before infusion may be a reasonable therapeutic to facilitate immune recovery and reduce treatment following

#{149}

±

autologous PSCT recipients based crease T cell functions. We suggest, CD14

12,592

0.409

digital autoradiographs of the Southern blots PBL from normal donors. ‘Twenty-four-hour PHA-stimulated normal Significantly different from normal PBL.

of

±

TNFbd

Clnhibitory

moval

154,873

PBLI 9

El.

d Ratio

cellular

PHA

±

40.8

Products

10

±

4:lb

NSC

Cell

N PBL

6

%a

such

by Stem

cancers

that

secrete

granulocyte/macrophage

colony-stimulating

factor.

Clin. Cancer Re.s. 1, 95-103. Young, M. R. I.. Young, M. E.. Wright, M. A. (1990) Stimulation of immunesuppressive bone marrow cells by colony-stimulating factors. Exp. Hemal. 18, 806-811. Palathumpat, V., Dejbakhsh-Jones, S., HoIm, B., Wang. H.. Liang, 0.. Strober, S. (1992) Studies of CD4CD8a3 bone marrow T cells with suppressor activity. J. ImmunoL 148, 373-380. Maier, T., Holda, J. H., Claman, H. N. (1986) Natural suppressor (NS) cells: Members ofthe LGL regulatory family. immunol. Today 7. 312-315. Oseroff, A., Okada, S., Strober, S. (1984) Natural suppressor (NS) cells found in the spleen of neonatal mice and adult mice given total lymphoid irradiation (TLI) express the null surface phenotype. J. ImmunoL 132, 101-110.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Mortani, F., Singhal, S. K. (1988) Production of human bone marrowderived suppressor factor. Effect on antibody synthesis and lectin-activated cell proliferation. J. Immunol. 141. 3037-3042. Maier, T., Holda, J. H., Claman. H. N. (1985) Graft-vs-host reactions (GVHR) across minor munine histocompatibility barriers. II. Development of natural suppressor cell activity. J. Immunol. 135, 1644-1651. Maier, T., Holda, J. H., Claman, H. N. (1989) Munine natural suppressor cells in the newborn, in bone marrow, and after cyclophosphamide: Genetic variations and dependence on IFNgammat. J Immunol. 143. 491 -498. Sykes. M.. Eisenthal, A., Sachs, D. H. (1988) Mechanism of protection from graft-vs-host disease in munine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component. J. Immunol. 140. 2903-2911.

29.

Mollereau, Bernard. A. (1996)

33.

B.. Deckert, M., Deas, 0.. Rieux-Laucat. F., Hirsch, F.. A.. Fischer. A., Lynch, H., Charpentier, R. LeDeist. F.. Senik. CD2-induced apoptosis in activated human peripheral T cells. J. Immunol. 156. 3184-3190. Szabolcs, P.. Avigan. D., Gezelter. S.. Ciocon. D. H.. Moore, M. A. S.. Steinman, R. M., Young, J. W. (1996) Dendnitic cells and macrophages can matune independently from a human bone marrow-derived. post-colonyforming unit intermediate. Blood 87, 4520-4530. Wanebo. H. J.. Riley. T.. Katz. D.. Pace. R. C.. Johns. M. E.. Cantrell, R. W. (1988) Indomethacin sensitive suppressor-cell activity in head and neck cancer patients. The role of the adherent mononuclear cell. Cancer 61, 462-474. Farmnas, M. C., Rodniguez-Valverde V.. Zarrabeitia, M. T., Parra-Blanco, J. A., Sanz-Ortiz, J. (1991) Contribution of monocytes to the decreased lymphoproliferative response to phytohemagglutinin in patients with lung cancer. Cancer 68, 1279-1284. Hertel-Wulif. B.. Strober, S. (1988) Immunosuppressive lymphokine denived from natural suppressor cells. J. Immunol. 140, 2633-2638. Vlasselaer, P. V., Niki, T., Strober, S. (1991) Identification of a factor(s) from cloned murine natural suppressor cells that inhibits IL-2 secretion during antigen-driven T cell activation. Cell. ImmunoL 138. 326-340.

Mills,

C. D. (1991)

metabolism

30.

31 .

via

Molecular the

basis

nitric

oxide

of “suppressor” synthetase

pathway.

macrophages. Arginine J. ImmunoL 146.

2719-2723. Schleifer, K. W., Mansfield, J. M. (1993) Suppressor macrophages in African trypanosomiasis inhibit T cell proliferative responses by nitric oxide and prostaglandins. J. ImmwsoL 151, 5492-5503. Tomioka, H.. Sato, K., Maw, W. W.. Saito, H. (1995) The role of tumor necrosis

factor,

interferon-y,

transforming

the expression of immunosuppressive duced by Mycobacterium avium

growth

factor-a.

and

nitric

oxide

functions of splenic macrophages complex infection. J. Leukoc. BiOL

in

in58.

704-712.

32.

34.

35.

36.

Munn, D. H.. Pressey, J., Beall. A. C.. Hudes. R.. Alderson, M. R. (1996) Selective activation-induced apoptosis of peripheral T cells imposed by macrophages. 1 Immunol. 156, 523-532. Wu, M. X.. Daley, J. F., Rasmussen, R. A., Schlossman, S. F. (1995) Monocytes are required to prime peripheral blood I cells to undergo apoptosis. Proc. Nazi. Acad. Sci. USA 92, 1525-1529. Wu. M. X., Ao Z., Hegen. M.. Monimoto, C., Schlosaman. S. F. (1996) Requirement of Fas(CD9S), CD4S, and CD11a/CD18 in monocyte-dependent apoptosis of human T Cells. J. Immunol. 157, 707-713. Palathumpat, V., Dejbakhsh-Jones. S., HoIm. B.. Strober, S. (1992) Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease. I ImmunoL 149, 808-817. Korbling, M.. Przepiorka, D., Huh, Y. 0., Engel. H., Van Besien. K., Giralt, S..

37.

38.

Anderson.

B..

Kleine,

H.

D..

Seong,

D.,

Deisseroth,

A.

R

(1995)

ADo-

geneic blood stem cell transplantation for refractory leukemia and lymphoma: Potential advantage of blood over marrow allografts. Blood 85, 1659-1665. Azevedo, W. M. (1995) Allogeneic transplantation with blood stem cells mobilized by rhG-CSF for hematological malignancies. Bone Marrow Traasplans. 16. 647-653. Liberti. C., Pearce, R., Taghipour. C.. Majolino, I., Goldsione, A. H. (1994) Comparison of peripheral blood stem-cell and autologous bone marrow transplantation

EBMT

mw

Registry

ci a!.

for

data.

CD14

lymphoma

Ann.

cells

patients:

OncoL

in

A case-controlled

analysis

of the

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mobilized

PSC

products

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