[CANCER RESEARCH55, 1099-1104, March 1, 1995]

Generation of Immunostimulatory Dendritic Cells from Human CD34+

Hematopoietic Progenitor Cells of the Bone Marrow and
Peripheral Blood'
Helga Bernhard, Mary L. Disis,2 Shelly Heimfeld, Susan Hand, Julie R. Gralow, and Martin A. Cheever
Department of Medicine, Division of Oncology, University of Washington, Seattle, Washington 98195 (H. B.. M. L D.. S. Ha.. J. R. G., M. A. C.). and CeliPro Inc., Bothell,
Washington 98021 (5. He.]

ABSTRACT
Dendritic antigen-presenting cells are considered to be the most effec
dye stimulators of T cell immunity. The use of dendritic cells has been
proposed to generate therapeutic T cell responses to tumor antigens in
cancer patients. One limitation Is that the number of dendritic cells in
peripheral blood Is exceedingly low. Dendritic cells originate from CD34@

For cancer therapy, investigators

have proposed

using DC pulsed

with tumor antigen as therapeutic vaccines in vivo, as well as priming

cancer antigen-specific T cells in vitro for use in adoptive T cell
therapy (35,10, 13). However, isolation of adequate numbers has
been difficult, because DC are present in the peripheral blood in very
low numbers (3). Caux et a!. (14) showed that DC could be derived
and expanded from CD34@ HPC in umbilical cord blood by inducing
dendritic cell differentiation and proliferation with GM-CSF plus

hematopoletic progenitor cells (HPC) which are present in the bone

marrow and in small numbers in peripheral blood. CD34@ HPC can be
mobilized Into the peripheral blood by in vivo administration
of granule
cyte-colony-stimulating
factor. The aim of the current study was to de

termine whether functional dendritic cells could be elicited and grown in

vitro from CD34@HPC derived from bone marrow or granulocyte-colony
stimulaflng factor-mobilized peripheral blood. Culture of CD34@ HPC
with granulocyte-macrophage-colony-stlmulating

capacity of DC to capture and process antigens, transport them to

lymph nodes, and therein activate naive T cells nominates them to a
specializedsubset of antigen-presenting cells, as suggested by
Steinman (3) and Knight and Stagg (10).

TNF-a (14). The current experiments asked whether DC could be

factor and tumor necro

typical dendritic morphology. Phenotypic studies demonstrated a loss of

the CD34 molecule over 1 week and an increase in cells expressing surface

similarly derived and expanded from CD34@ HPC in bone marrow or

peripheral blood of cancer patients. CD34@ HPC are present in the
peripheral blood in only low numbers, comprising less than 1% of the

markers

WBCs. However, the enrichment and isolation of CD34@stem cells

515 factor

a yielded

a heterogeneous

associated

cell population

with dendritic

cells, CD1a,

containing

cells with

CDSO (B7IBB1),

CD4,

CD14, HLA-DR, and CD64 (FciRI). Function was validated in experi

meats showing that CUltUredcells could stimulate proliferation of alloge
neic CD4' and CD8@T lymphocytes. Antigen-presenting capacity was
further confirmed in experiments showing that cultured cells could effec
tively stimulate tetanus toxoid-specific responses and HER-2/neu peptide
specific responses. The derivation and expansion of dendritic cells from
cultured bone marrow or granulocyte-colony-stimulating
factor-mobi
lized CD34+

HPC

may provide

adequate

numbers

for testing

of dendritic

cells in clinical studies, such as vaccine and T cell therapy trials.

INTRODUCTION
DC3 are APC that are critical for the initiation of T cell responses
in vivo including sensitization of MHC-restricted T cells and devel
opment of T cell-dependent antibodies (17).DC originate from
@J@34+pluripotent HPC in the bone marrow and migrate as immature
cells to nonlymphoid tissues such as skin (Langerhans cells), mucosa,
and tumor (1, 8, 9). During antigen-induced immune responses, DC
take up antigen, migrate through the afferent lymphatic system to the
lymphoid organs, and efficiently present antigen to T cells (3). DC are
thought to be the major APC type involved in triggering primary T
cell responses (10). A number of studies have demonstrated that
human DC derived

from the peripheral

than peripheral blood-derived

blood are more potent APC

18 U.S.C. Section 1734 solely to indicate this fact.

work

has

been

supported

by

Deutsche

Forschungsgemeinschaft

Berlex

Oncology

Foundation

Fellow.

as well

as with a peptide

derived

from the normal

the development of dendritic cell-based vaccines and T cell therapy

To

whom

requests

for

reprints

should

be

addressed, at Division of Oncology (Mailstop: RM-17), 1959 Northeast Pacific Street,

Health Science Building BB1321, Seattle, WA 98195.
3 The abbreviations used are: DC, dendritic cells; APC, antigen-presenting

MATERIALS

AND METHODS

Source of Cells. Peripheralblood mononuclearcells were obtainedfrom

breast cancer patients as well as from healthy donors, bone marrow samples

Grant

Be1579/11 (H. B.) and NIH Grants ROl CA57851 and 5 ROl CA49850 (S. H., J. R. G.,
M. A. C.).
2

protein

amino acid sequence of HER-2/neu. This oncogenic protein is over

expressed in adenocarcinomas of different origins, such as breast
cancer, and is a potential target for immunotherapy (1518).The
demonstrated ability to procure increased numbers of DC might allow
regimens.

Received 9/6/94; accepted 1/3/95.

Pals

toxoid

monocytes or B cells (11, 12). The

Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page

charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith
1

is now a standard procedure for stem cell support following high-dose

chemotherapy. For our peripheral blood studies adequate numbers of
CD34@ cells could be positively selected by antibody affinity col
umns after stem cell mobilization by in vivo administration of G-CSF
for 3 to 4 days followed by leukapheresis.
The results demonstrated that culturing CD34@ HPC, which were
derived either from BM or PBSC collections, with GM-CSF plus
TNF-a induced differentiation of heterogeneous cell populations in
cluding cells expressing dendritic cell morphology and phenotype.
The cultures containing DC were able to stimulate proliferation of
allogeneic T cells in vitro, the most common assay used to evaluate
dendritic cell function. Critical to the development of a vaccine
strategy, the cultured DC should also function as APC for the stim
ulation of T cell responses to protein and peptides. Autologous T cell
proliferation could be induced by loading DC with whole tetanus

cells; HPC,

hematopoietic progenitor cells; PBSC, peripheral blood stem cells; BM, bone marrow;
G-ChF, granulocyte-colony-stimulatingfactor;GM-CSF,granulocyte-macrophage-colo
ny-stimulating factor; FACS, fluorescence-activated cell sorting; TNF-a, tumor necrosis
factor a; MLR, mixed leukocyte reaction; PE, phycoerythrin.

fromhealthydonorsonly.PBSCwerecollectedfrompatientsaboutto undergo
autologous stem cell transplantation for breast cancer. The majority of the
patients had advanced stage breast cancer in complete or partial remission with
stable clinical condition at the time of collection. Patients were not actively
receiving chemotherapy. Briefly, patients were administered G-CSF at a dose

of 10 pg/kg daily for 3 to 4 days to mobilize stem cells. Leukapheresis was

performed on day 3 or 4. Aliquots (3 ml) of the leukapheresis product were
used to purify CD34@HPC.

1099

GENERATION OF IMMUNOSTIMULATORYDENDRITIC CELLS

Separation
of CD34@ HPC and CD4@ or CD8@ T Lymphocytes.
CD34@ HPC were isolated from bone marrow or G-CSF-mobilized peripheral
blood. CD4@ and CD8@ T lymphocytes

were used as responder cells. Dendritic cell cultures were harvested after
1216days, irradiated with 30 Gy, and added to the responder cells at different

were purified from peripheral blood

concentrations

mononuclear cells or PBSC using the cell separation system Ceprate LC Kit
(CelIPro, Bothell, WA; Ref. 19). First, samples were processed using Ficoll
Hypaque density gradient centrifugation (Pharmacia, Piscataway, NJ). Cells
obtained from the interface were washed and resuspended in PBS-1% BSA. In
order to purify CD34@ HPC, up to 108 cells/mI were incubated for 25 mm on
ice with 40 @g'mlbiotinylated mAb anti-CD34 in PBS-1% BSA. Purification
of CD4@ and CD8@ I lymphocytes required a two-step labeling using 20
@g'mlanti-CD8 mAb or anti-CD4 mAb, respectively, for 25 mm on ice. Cells
were washed with PBS-l% BSA and incubated with a biotinylated antimouse

(102-104/well).

The assay

was performed

in 96-well

round

bottomed plates (Corning, Coming, NY) at 37C.The medium used consisted

of equal parts of EHAA (Biofluids) and RPMI 1640 (Gibco) with 10 mM
L-glutamine, 200 units/ml penicillin, 200 units/ml streptomycin, 2.5 X iO@ M
2-mercaptoethanol, and 10% human serum (human AR CELLect; ICN Flow,

Costa Mesa, CA). After 4 days, cells were pulsed with 50 pJ/well [3H]thymi

1 ml PBS-5% BSA/lO' cells, was applied to the Ceprate column, which

contained sterile avidin-coated polyacrylamide beads. After washing with

dine (1 mCi/rn]) for 8 h and counted. Data are shown as the mean of four
replicates.
Antigen-presenting
Assay. For induction of antigen-specific T cell re
sponses two different antigens were used. Peptide p4256,derived from the
amino acid sequence of HER-2/neu protein, is 15 amino acids in length and
was chosen based on an increased probability of binding to Class II MHC
molecules (17). The peptide was synthesized and purified by Dr. P. S. H. Chou
(University of Washington, Seattle, WA). Preservative-free tetanus toxoid was

PBS, the attached cells were removed from the beads by mechanical agitation

chosen as a representative protein antigen (Wyeth).

mAb for an additional 25 mm. The biotinylated cells were washed with
PBS-1% BSA to remove any unbound antibody. This fraction, in a volume of

and eluted with PBS.

Purity of recovered

Dendritic

by staining with anti-CD4 and anti-CD8 mAb, respectively. The selected I

cells were not activated through the selection procedure. Purity of CD344 cells
depended on the source of the HPC. CD34@ cells isolated from bone marrow
regularly showed a purity of 80% to >95%, whereas the purity from PBSC
derived CD34@ cells ranged from 30 to 80% depending on the response to

G-CSF treatment of the individual patient.

Dendritic Cell Culture Derived from HPC. DC were generatedusing a
modified method, described by Caux et al. (14), for generating DC from
umbilical cord blood. All cell cultures were initiated from CD34@ HPC, which
had been isolated through positive selection by immunoaffinity columns. The
purified cells were resuspended in RPM] 1640 medium (GIBCO, Grand Island,
NY) containing

2.5 X i0@

mM 2-mercaptoethanol,

100 units/mI

penicillin,

100 @Wmlstreptomycin, 2 mM L-glutamine, and 10% FCS. The culture

medium was further supplemented with GM-CSF (100 ng/ml) (Schering
Plough, Kenilworth, NJ) and INF-a (2.5 ng/ml) (Genzyme Corp., Cambridge,

MA). CD34@HPC were plated into 24-well plates (Costar, Cambridge, MA)
at a final concentration of i0@ cells/ml/well and split every 45days. After
1216days of culture time, cells were harvested and used for phenotyping and
functional assays.
Monocyte Culture. Monocytes were enriched as described previously (2).
In short, PBSC (2 x l06/ml) were cultured in supplemented RPMI 1640 in
Petri dishes (100 mm; Falcon, Lincoln Park, NJ). After 36 h of culture at 37C,
plates were washed three times in order to remove the nonadherent
cells. Cells

that remained adherent or that readhered to tissue culture plastic after 36 h

were used as an enriched
adherent

cells displayed

source of monocytes.
the monocyte

marker

In FACS analysis,

cell cultures

were

harvested,

spun,

amounts of EHAA and RPMI 1640 supplemented

CD4@ and CD8@ cells was 90% to >95% as determined

70% of the

CD14.

Flow Cytometric Analysis. Ihe following mAbs were used for FACS
analyses: anti-CD1a, anti-CD34 (Becton Dickinson, San Jose, CA); anti-CD8O
(B7/BB1) (provided by Dr. E. Clark, University of Washington, Seattle, WA);
anti-CD35 (complement receptor CR1; ACCU, Westbury, NY); anti-CD64
(FcyRI; Medarex, Annandale, NJ), PE-conjugated anti-CD1a, anti-CD3, anti

and resuspended

in equal

as described above. Differ

ent cell concentrations were loaded with tetanus toxoid protein (25 p@g/ml)
or
HER-2/neu

p4256 peptide

(50

@g/ml). Following

an incubation

period for 1

to 2 h at 37C,the pulsed dendritic cell population was irradiated (30 Gy) and
added to autologous CD4@ I lymphocytes that acted as responder cells

(5 X 104/well).Proliferativeresponsewas measuredas a functionof thymidine

uptake and results are shown as the mean of four replicates.
RESULTS

DC Can Be Generated from CD34@ HPC from BM and Pe

ripheral Blood in the Presence of GM-CSF and TNF-a. Initial
experiments asked whether the methods developed by Caux et a!. (14)
for growing DC from cord blood could induce the growth of DC from
adult bone marrow. CD34@ HPC from BM were enriched by positive
selection (90% purity) and cultured with GM-CSF plus TNF-a for 12
to 16 days. Results showed that the total cell number increased
approximately 40-fold, ranging from 20- to 80-fold (Fig. 1A). During
cell growth the CD34@ HPC population lost the CD34 marker which
was undetectable by day 9, and cells with a more differentiated
morphology

appeared

(Fig. 1B). In order to follow

the differentiation

of CD34@ HPC into DC, the CD1a molecule, known as a marker for
dendritic Langerhans cells, was used. CD1a expression gradually
increased, reaching a maximum level on day 15 (20). At that time
point the cultured population was heterogeneous, and the CD1a mol
ecule was displayed by 3060% of the cells, depending on the
individual cell culture. The relative number of CD1a expressing cells
200

100AB0

CD4, anti-CD8, anti-CD56, anti-CD19 (Becton Dickinson, San Jose, CA);

anti-HLA-DR-PE (Olympus Corp., Lake Success, NY), FIIC-F(ab')2 frag
ment goat anti-mouse IgG (Zymed, San Francisco, CA).
Double-color fluorescence staining of cell cultures was carried out by
sequential incubation of mAbs. Briefly, cells were resuspended in PBS-1%

2E

at saturating

concentrations

for 10 mm on ice, washed

Negative controls were performed with FITC-F(ab')2 goat anti-mouse IgG and
a PE-conjugated unrelated murine mAb (Becton Dickinson). Fluorescence
with a FACScan

flow cytometer

(Coulter).

MLR. Naive CD4@ or CD8@ I lymphocytes (5 X 104/well) isolated

through

the Ceprate

LC Kit from normal

donor peripheral

blood lymphocytes

20

0@
1

Finally, cells were incubated with PE-labeled mAb for 20 mm,

were performed

at6

0
5

11

15

Days in Culture

washed twice with PBS-1% BSA and fixed with 1% paraformaldehyde.

analyses

a@ 40
50

PBS-1% BSA, 10 pi mouse immunoglobulinswere added for 10 mm to reduce

>

twice

CD34

:n
0)

with 150 @.d

PBS-l% BSA and stained with FIIC-F(ab')2 fragment conjugated
goat anti-mouse IgG for an additional 20 mm. Following two washes with
cross-reactivity.

100

In order to reduce nonspecific binding, 10 @l

goat immunoglobulin (3 mg/mI)
were added to each well. After 10 mm, cells were incubated with the uncon
mAbs

.-COla
B

BSA and seeded into microtiter plates at a final concentration of iO@cells/well.

jugated

80

1 50
0

19

11

15

19

Days in Culture

Fig. 1. Kinetics (A) and phenotype (B) of dendritic cell generation from BM-derivcd
CD34@HPC cultured with GM-CSF and TNF-co.Expansion of the CD34@HPC starting
population during a 19-day culture period is shown. CD34@HPC lost the CD34 marker
during cell growth. Expression of CD1a, a marker related to DC, increased during culture,
reaching a maximum level on day 15. Decreasing CD1a expression after day 15 is related
to the expanding monocytes.

1100

@
@

GENERATION OF IMMUNOSTIMULATORYDENDRITIC CELLS

molecule (Fig. 4). All of the CD1a-positive cells were also positive for
the activation molecule CD8O (B7IBB1). The majority of CD1a@ cells
(8090%)were positive for CD4, which is known to be expressed by
cultured DC (20). Subpopulations of CD1a@ cells (3050%) were
positive
@

@iyJ.

for the monocyte

marker

CD14,

which

indicates

that the

CD1a@ population can subdivide into CD1a@/CD14 cells and

CD1a@/CD14@ cells.
Allogeneic T Lymphocyte Proliferation Can Be Induced by DC
Generated from CD34@ HPC. Cultured cell populations containing
DC were tested for the capacity to induce proliferation of allogeneic
T cells. After 1214days of culture, cells generated from BM or
mobilized peripheral blood HPC-derived CD34@ HPC were used as

@f>

B7/BB1

CD4

CD1a

Fig. 2. Morphology of adherent cells generated from CD34@ HPC in response to

GM-ChFand1'NF-a examinedunderphase-contrastmicroscopy.Adherentcells display
a dendritic morphology with delicate membrane projections. X 200.

@/J \j

decreased after day 15 of culture due to expansion of cells with

monocyte and macrophage morphology. In the experiment presented,

15 X 10@CD1a@cellswere derivedfrom a startingpopulationof

@

@
@

@
@
@

2 X 10@CD34@ HPC.
Similar growth was observed for both BM- and peripheral blood
derived CD34@ HPC. The amount of cell growth strongly correlated
with the degree of purity of the starting CD34@ cell population. This
observation was documented in 22 independent cell cultures. Periph
era] blood-derived CD34@ HPC were, in general, less pure (3080%)
than BM-derived HPC (80 to >95%) after positive selection and
expanded to a lesser degree (520-fold)proportional to the percentage
of CD34@ cells in the initial culture.
Cultured Cells Derived from CD34@ HPC Display Heterogene
ous Morphology and Phenotypic Markers of DendriticlLanger
hans Cells and Monocyte/Macrophages
Including the Expression

HLA-DR

L.J'@'\:@@@
c03

C035

and 15 with BM- and peripheral blood-derived

@D8

/@:\

of Class II MHC and Costimulator CD8O (B7IBB1) Molecules.

The morphology of cultured cells was evaluated at days 1214.
During 2 weeks of culture the homogeneous population of small
mononuclear cells differentiated into a heterogeneous population of
adherent and nonadherent cells which could be distinguished by
phase-contrast microscopy as three major cell populations. Approxi
mately 50% of the total cells were adherent or loosely adherent, large
and vacuolated, with or without a few short membrane projections,
typical features of monocytes and macrophages. The second major
cell population consisted of small, round, and nonadherent cells
without vacuoles or projections which represented about 30% of the
cell culture. This morphologically defined second cell population
could not be categorized as a known functionally differentiated cell
type. The third and smallest population (20%) contained mostly
adherent cells with a dendritic cell morphology with long delicate
membrane projections (Fig. 2). The cells with dendritic morphology
were generally overgrown by typical macrophages after 2 weeks.
Immunofluorescence analyses were performed between days 12

CD64

CD14

I}
@L-@

\\

I
@L-:-@

@-_@--

C056

C019

CD34

I \

L/@@
Fig. 3. Cell surface phenotype of BM- and peripheral blood-derived CD34@ cells
cultured for 2 weeks in the presence of GM-CSF and TNF-a. Dashed lines, stainings with
isotype-matched nonreactive control mAb; bold lines, staining with mAb as indicated. The
cell cultures were positive for CD1a, CD4, CD8O(B7/BBI), HLA-DR (Class II MHC),
CDI4, CD64 (Fc'yRI),and CD35 (complement receptor CR1) and were negative for CD3,
CD8, CD19, CD56, and
DCSurface Table 1Phenotype of HPC-derived

DCCD1al0-.60@'CD410-90CD1420-80CD8O10-60HLA-DR50-90CD34<5CD3<5CD8<5C
antigensHPC-derived

dendritic cell cultures.

Representative FACS analyses are presented in Fig. 3. The cultured

cells did not display cell surface markers for B lymphocytes (CD19),
T cells (CD3, CD8), or natural killer cells (CDS6). Surface markers
expressed by the heterogeneous cultured populations included CD1a,
CD4, CD14, CD8O (B7/BB1), HLA-DR (Class II MHC), CD64
(FcyRI), and CD35 (complement receptor CR1). The expression of
these markers varied from culture to culture, as shown in Table 1.
Results were similar for populations generated from BM or peripheral

bloodHPC.
To characterize the cell populations in more detail, double-color
flow cytometric analyses were performed using CD1a as a reference

a Cell

surface

phenotype

of

BM

and

peripheral

blood-derived

CD34@

cells

varied

between cultures. The range of cell surface marker expression for CD1a, CD4, CD14,

CD8O,and HLA-DR is shown. All other markers were reproducibly negative. High levels
of expression of CD1a and CD8Owere routinely found in cultures of the highest initial
CD34 purity.
b Percentage of positive cells.

1101

GENERA11ON
OF IMMUNOS11MULATORY
DENDRI11CCELLS

8000

stimulator cells for allogeneic CD4@ as well as CD8@ T lymphocytes

that had been isolated using positive selection (>95% purity). Den
dritic cell cultures derived from either BM (4/4) or PBSC (6/6)
induced a strong proliferative T cell response for both CD4@ and
CD8@ T lymphocytes, as demonstrated by an increased thymidine
incorporation (Fig. 5). The number of T cells/well was held constant
and the number of cultured DC as stimulator cells varied. The level of
T cell proliferation was dependent on the number of DC/well. The T
cells were not observed to be activated by the method of purification
used, as determined by a lack of thymidine incorporation without
stimulator cells added.

#@

0@

C-)

a,

I 4000@

DC Generated from CD344 HPC Can Present Antigens to

Autologous CD4@ T Lymphocytes. The ability of positively se
lected CD4@ T cells from patients with breast cancer to respond to
protein and peptide antigens presented was examined in five individ
uals. Autologous CD4@ T lymphocytes purified from frozen PBSC
acted as responder cells and PBSC-derived dendritic cell cultures as
APC. Soluble protein antigen or synthetic peptide was added to
variable numbers of cultured DC with a fixed number of autologous
CD4@ cells (Fig. 6). Tetanus toxoid was used as a representative recall
protein. The peptide used, denoted as HER-2/neu p4256,was a
15-amino acid peptide derived from the normal sequence of HER-2/
neu, a protein which is overexpressed in many breast cancer patients.
As the number of DC/well increased, the proliferative response of
CD4@ T lymphocytes to tetanus toxoid and HER-2/neu peptide was
observed to increase. In this patient, the response to tetanus toxoid
was greater than the response to HER-2/neu peptide. Of note, DC
alone at higher numbers induced a T cell response presumably re
flecting an autologous MLR. In a second patient, the capacity of DC
generated from CD34@ HPC to present peptides was compared to

6000

2000@

0@
0

Number

10

of stimulator

cells per well

Fig. 6. Antigen presentation by dendritic cell cultures. Antigen-pulsed PBSC-dcrlved

DC from a breast cancer patient induced a protein- or peptide-specific CD4@response.
HER-2/neu p4256peptide,

@,
tetanus toxoid protein, t

the five patients tested, a proliferative

B7/BB1

no antigen, A.

CD1a

COla

10000

0.
U

1000

0.
.@

100

10

100 1000100000

Number of stimulator

10

100 100010000

cells per well

Fig. 5. MLR using BM-derived dendritic cell cultures as stimulator cells and aliogeneic
CD4@and CD8@T lymphocytes as responder cells. T lymphocyte proliferation can be
induced by DC, which were generated from CD34@HPC with GM-CSF and TNF-a.
DC cultures derived from either BM (4/4) or PBSC (6/6) are capable of inducing
alloreactivity.

could

DISCUSSION

number equivalent

to HER-2/neu

Developing the immunogenic potential of DC for cancer therapy

requires better access to this rare cell type (3). This report demon
strates that DC can be derived from positively selected CD34@ HPC.
Culturing positively selected CD34@ HPC with TNF-a and GM-CSF
resulted in an approximately 40-fold expansion of the starting cell
number over 2 weeks and the generation of CD34-negative DC in a

CD4

response

not be detected.

Fig. 4. Double-color flow cytometric analysis of BM-derived CD1a cells. All CD1a@
cells express CD8O (B7IBB1). The majority of CD1a@ cells were CD4 positive. A
subpopulation of CDIa cells was also positive for CD14.

a,

1000 10000

monocytes. As shown in Fig. 7, peptide-pulsed DC induced autolo

gous CD4@ T cells to proliferate. In comparison, monocytes from the
same patient were not able to induce autologous CD4@ T cells to
proliferate. Again, DC alone stimulated T cell proliferation. In three of

.@
(5

100

to at least the number of starting CD34@ cells.

CD34@ HPC derived from either G-CSF-mobiized peripheral blood

or BM are capable of acting as the starting population. The advantages
of using BM over PBSC are a greater purity of CD34@ stem cells
following cell separation (80 to >95% versus 3080%), and subse
quent better cell growth (2080-fold versus 520-fold). The col
lection of PBSC results in a higher total stem cell number after
leukapheresis. Since the purity of CD34@ stem cell preparations
varied from donor and source, it cannot be formally ruled out that DC
originated from CD34-negative DC progenitors. However, the stron
ger growth of highly pure CD34@ from the BM compared to low
purity CD34 preparations from the peripheral blood indicates that DC
cultures are derived mainly from CD34@ cells. This hypothesis is
supported by single-cell experiments, which revealed that DC cob
nies originate from CD34@ cells of the BM, as recently reported by
Reid et aL (21, 22).
The rationale for using TNF-cs in addition to GM-CSF was drawn

1102

GENERATION

OF IMMUNOSTIMULATORY

5000

I 4000
0@
C-,
-@
4@)

0@
@0

F-

2000

1000@

Dendritic cells

Monocytes

Fig. 7. Comparison of dendritic cell cultures and monocytes, from a breast cancer
patient, as APC. DC and monocytes were loaded with HER-2/neu p4256(R) or no
antigen (Cl). Peptide-pulsed PBSC-derived DC induced a specific CD4@ response to

HER-2/neu p4256
peptide, whereas monocytes from the same patient were not able to
present the peptide to the autologous @T@+
T lymphocytes.

from the experience of others who showed that TNF-a modifies

GM-CSF-induced myelopoiesis and facilitates the development of DC
from cord blood CD34@ HPC (14). Using this regimen to culture
positively selected CD34@ HPC from BM or peripheral blood resulted
in a heterogeneous population of three major cell types based on
morphology: DC, monocytes, and small round cells of unknown
function. The DC were mostly adherent and displayed a typical
morphology with delicate membrane projections. The monocytes
were largely adherent and vacuolated and formed a tight cell network
which tended to overgrow the DC after 2 weeks. Both DC and
monocytes were expanded under the same conditions. It is possible
that they may share the same precursor, a concept recently presented
by Reid et a!. (21, 22), and is supported by the observation that fully
differentiated monocytes could be a source for generating veiled
accessory cells when cultured in serum-free medium (23, 24). Future
attempts at generating DC from CD34@ HPC should explore the
influence of other cytokines to drive HPC differentiation into a more
homogeneous population of DC. Cytokines of interest include inter
leukin 1 which signals interleukin 1 receptor to up-regulate GM-CSF
receptors on epidermal DC; interleukin 4 which drives monocytes into
cells with dendritic processes, down-regulates monocyte markers, and
enhances antigen-presenting capacity; and interleukin 6 which stim
ulates monocyte-derived Langerhans cells via autocrine cytokine
production (2428).
The cell populations derived by culture of CD34@ HPC lost the
CD34@ marker, were positive for markers known to be expressed by
DC and monocytes (CD1a, CD4, CD8O, and Class II MHC; Refs. 3,
14, 2933),and lacked surface markers for B, T, and natural killer
cells (CD3, CD19, CD8, and CD56). The studies focused particularly
on the CD1a molecule which is associated with the immunostimula
tory cell fraction from HPC-derived cell cultures (14). Double-color
immunofluorescence showed that the majority of CD1a@ cells ex

DENDRITIC

CELLS

pressed CD4 and CD8O (B7/BB1). Approximately one half of the

CD1a@ cells expressed CD14, a monocytic marker. It is not clear
whether only the CD1a@/CD14 cells should be designated as den
dritic cellsor whether some DC are CD1a@/CD14@. Others have
shown that DC can be sorted from dimly stained CD14@ cells (34).
An important issue not addressed is possible functional differences in
antigen presentation between the defined phenotypic populations.
Function of cultured DC was confirmed by examining their ability.
to induce proliferation of resting allogeneic CD4@ and CD8@ T
lymphocytes (2, 3, 14, 35). Capacity to induce strong alloreactivity
was consistently shown using DC cultures derived from either bone
marrow (4/4) or mobilized peripheral blood HPC (6/6). In contrast,
the ability to stimulate protein- and peptide-specific responses was
less consistent. In the experiments presented, cultured DC effectively
stimulated tetanus toxoid-specific responses and HER-2/neu peptide
specific responses (2/5). However, some dendritic cell cultures stim
ulated strong allogeneic responses, but not protein- and peptide
specific responses (3/5). The lack of a detectable antigen response
might be due to a low precursor T cell frequency in those patients.
Another reason for the preferential ability of DC to stimulate alloge
neic responses might be a down-regulation of the ability to present
exogenous antigen after maturation in culture. It is known that intact
protein is presented best by immature DC (5, 27, 36, 37). A major
issue that remains is identifying culture conditions that expand DC
which consistently retain the ability to process and present exogenous
antigens. Other studies, as well as the data presented here, demon
strate augmentation of MLR with human DC derived from progenitor
cells (14, 38). Results from our laboratory, however, show that cx
trapolation of dendritic cell function from MLR to specific antigen
presenting capability is not necessarily appropriate. Although function
in a MLR is reproducible, these same DC would have variable
function when presenting specific antigens. Subsequent studies should
focus on the interrelationship between cell growth and the functional
ability to stimulate protein- and peptide-specific responses and should
utilize antigen-presenting function rather than cell number or alloge
neic MLR for optimization of culture conditions. The availability of
sufficient numbers of efficient antigen-presenting DC should facilitate
the study of T cell-mediated responses to tumor-associated antigens.
ACKNOWLEDGMENTS
We thank Ed Clark for providing anti-CD8OmAb B7/BB1, Kirsten Stray,
Sandra R. Emery, and Faith Shiota for technical assistance, and Kevin
Whitham for manuscript preparation. In addition, we thank the members of the
autologous bone marrow transplant team, leukapheresis unit, and cryopreser
vation unit at the Fred Hutchinson Cancer Research Center for supplying the

peripheral blood stem cells used in these studies.

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