Dendritic Cell-based Vaccines in the Setting of Peripheral ... · Dendritic Cell-based Vaccines in...

Vol. 4, 2709-2716, November 1998 Clinical Cancer Research 2709

Dendritic Cell-based Vaccines in the Setting of Peripheral Blood

Stem Cell Transplantation: CD34� Cell-depleted Mobilized

Peripheral Blood Can Serve as a Source of Potent

Dendritic Cells1

David Choi, Michelle Perrin, Sandra Hoffmann,

Alfred E. Chang, Voravit Ratanatharathorn,

Joseph Uberti, Kevin T. McDonagh, and

James J. Mule2Departments of Surgery [D. C., A. E. C., J. J. M.] and InternalMedicine [V. R., J. U., K. i. MI, the Blood Bank IS. H.], and the

General Clinical Research Center [M. P.], University of MichiganMedical Center, Ann Arbor, Michigan 48 109-0666

ABSTRACTWe are investigating the use of tumor-pulsed dendritic

cell (DC)-based vaccines in the treatment of patients with

advanced cancer. In the current study, we evaluated the

feasibility of obtaining both CD34� hematopoietic stem/

progenitor cells (HSCs) and functional DCs from the same

leukapheresis collection in adequate numbers for both pe-

ripheral blood stem cell transplantation (PBSCT) and im-

munization purposes, respectively. Leukapheresis collec-

tions of mobilized peripheral blood mononuclear cells

(PBMCs) were obtained from normal donors receiving

granulocyte colony-stimulating factor (G-CSF) (for alloge.

neic PBSCT) and from intermediate grade non-Hodgkin’s

lymphoma or multiple myeloma patients receiving cyclo-

phosphamide plus G-CSF (for autologous PBSCT). High

enrichment of CD34�’ HSCs was obtained using an immu-

nomagnetic bead cell separation device. After separation,

the negative fraction of mobilized PBMCs from normal

donors and cancer patients contained undetectable levels of

CD34� HSCs by flow cytometry. This fraction of cells was

then subjected to plastic adherence, and the adherent cells

were cultured for 7 days in GM-CSF (100 ng/ml) and inter-

leukin 4 (50 nglml) followed by an additional 7 days in

GM-CSF, interleukin 4, and tumor necrosis factor a (10

ng/ml) to generate DCs. Harvested DCs represented yields of

4.1 ± 1.4 and 5.8 ± 5.4% of the initial cells plated from the

CD34� cell-depleted mobilized PBMCs of normal donors

and cancer patients, respectively, and displayed a high level

expression of CD8O, CD86, HLA-DR, and CD1 ic but not

CD14. This phenotypic profile was similar to that of DCsderived from non-CD34� cell-depleted mobilized PBMCs.DCs generated from CD34� cell-depleted mobilized PBMCs

elicited potent antitetanus as well as primary allogeneic

T-cell proliferative responses in vitro, which were equivalent

to DCs derived from non-CD34� cell-depleted mobilized

PBMCs. Collectively, these results demonstrate the feasibil-

ity of obtaining both DCs and CD34� HSCs from the same

leukapheresis collection from G-CSF-primed normal donors

and cancer patients in sufficient numbers for the purpose of

combined PBSCT and immunization strategies.

INTRODUCTIONDCs3 are highly potent APCs of BM origin (I ). which have

been shown to stimulate both primary and secondary T- and

B-cell responses (2, 3). We (4-7) and others (8-10) have shown

that tumor-pulsed DC-rich preparations can stimulate specific

T-cell reactivity in vitro and serve as potent antitumor vaccines

in VitO. Moreover, methods are now available to generate siz-

able numbers of highly enriched DCs in both rodents and

humans by culturing progenitor cells in the presence of GM-

CSF, TNF-a, and/or IL-4 ( 1 1-1 3). The establishment of DC

cultures from the peripheral blood of adult patients has raised

the very important possibility of now using these cells as an

immunotherapeutic agent for the treatment of a variety of hu-

man tumors (9, 14, 15).

Another form of adoptive immunotherapy involves the use

of BMT or PBSCT. This approach is currently being used for

the treatment of patients with cancer, including those with

lymphorna, leukemia, and metastatic breast cancer. Well-estab-

lished models exist in mice of successful transplantation and

hernatolymphoid reconstitution with whole, unfractionated BM

or with highly selected HSCs (16, 17). Investigators have also

Received 4/13/98; revised 8/20/98: accepted 8/26/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

‘ Supported by NIH Grants 1 P01 CA59327 and M01-RR00042 (to the

University of Michigan General Clinical Research Center), Department

of DefenseIUSAMRMC Grant DAMD17-96-l-6l03, and United StatesArmy Research Office Grant DAAG55-97-l-0239.2 To whom requests for reprints should be addressed, at the Departmentof Surgery, University of Michigan Medical Center, 1520c MSRB-l,1 150 West Medical Center Drive, Ann Arbor. MI 48109-0666. Phone:(734) 647-2779; Fax: (734) 763-4135.

3 The abbreviations used are: DC, dendritic cell; HSC, hematopoieticstem/progenitor cell; PBSCT, peripheral blood stem cell transplantation:

NHL, non-Hodgkin’s lymphoma; PBMC, peripheral blood mononuclearcell; G-CSF, granulocyte colony-stimulating factor; GM-CSF. granulo-

cyte macrophage colony-stimulating factor: TNF, tumor necrosis factor:

IL, interleukin; BM, bone marrow; BMT, BM transplant: APC. antigen-presenting cell: CM, complete medium; Ti’. tetanus toxoid: FACS.

fluorescence-activated cell-sorting; dThd, thymidine: PHA, phytohem-

agglutinin: MLR. mixed leukocyte response: mAb, monoclonal anti-

body; PE, phycoerythrin.

Research. on September 29, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

2710 DC-based Tumor Vaccines and PBSCT

cornbined the adoptive transfer of lymphokine-activated killer

cells and/or IL-2 with BMT or PBSCT, which has resulted in

potent antiturnor activity against residual disease in both the

mouse (18) and human (19). These promising results have

engendered much enthusiasm for strategies that would enhance

antitumor immune reactivity after BMTIPBSCT, yet they also

raise the possibility of further improvement.

Recent evidence suggests that thymic-independent T-cell

regeneration can occur via antigen-driven expansion of periph-

eral T cells. Important new studies have shown that active

immunization of the BM donor before transplant has resulted in

a strategy for enhancing the specific antiturnor effect of marrow

grafts. presumably as a result of early lymphoid progenitors

being prone to biasing and, as a result, generating a T-ceIl

repertoire that is limited in diversity (20-22). This approach

may arm a BMT or PBSCT graft with antitumor function,

provides a rationale for immunizing donors to optimize the

adoptive transfer of protective T-cell immunity into recipients,

and suggests the possibility of using preventive T-cell adoptive

therapy in conjunction with marrow or mobilized peripheral

blood transplantation. In addition, the possibility remains that

active immunization of the BMT recipient during reconstitution

may also result in enhanced antitumor immunity and bias the

developing T-cell repertoire (20-22). In this regard, murine

studies have shown that antigen-pulsed DCs can specifically

prime developing neonatal T cells and break immune tolerance

(23).

The potential of combining DC-based vaccines with

PBSCT is currently being considered based on the rationale that,

in addition to the above considerations, immunization with DCs

may have greater potential after transplant preparative regimens

that reduce or eliminate active tumor-induced immunosuppres-

sion and lessen tumor burden. Because of the potent APC

activity of DCs, potential exists to bias the developing immune

T-cell repertoire toward higher frequency recognition of tumor-

associated antigens through early immunization with tumor an-

tigen(s)-pulsed DCs after PBSCT. In the current study, we

demonstrate the feasibility of obtaining both DCs and CD34�

HSCs with potent functional activity from the same leukaphere-

sis collection from G-CSF-pnimed normal donors and cancer

patients in sufficient numbers for the purpose of combining

PBSCT with DC-based immunization strategies for the treat-

ment of cancer.

MATERIALS AND METHODSCulture Medium, Reagents, and Cytokines. The me-

dium used for DC cultures was serum-free XVIVO-15 (Bio-

Whittaker, Gaithersburg, MD). CM used for T-cell isolations

(see below) consisted of RPMI 1640 supplemented with 10%

heat-inactivated FCS, 100 units/ml penicillin, 100 p,g/rnl strep-

tomycin, 50 �i.g/ml gentamicin sulfate, 2 �i.g/m1 fungizone, 0.05

mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 0. 1 msi

nonessential amino acids.

Preservative-free IT was purchased as a sterile liquid in

vials from Connaught Laboratories, Ltd. (North York, Ontario,

Canada). The activity of IT was 2250 Lf/l2.2 mg/mi (lot

number TAS 319 R8), which was diluted further in XVIVO-15

to achieve 225 Lf/ml and stored at 4#{176}Cbefore use.

Recombinant human IL-4 was kindly provided by Scher-

ing-Plough Research Institute (Kenilworth, NJ). The specific

activity was determined to be 6.35 X l0� lU/mg (lot number

3-ENP-803). Vials were diluted with XVIVO-15 to 50 p.g/ml

and stored at -80#{176}Cbefore use.

Recombinant human GM-CSF was kindly provided by Dr.

C. Reynolds (Biologic Response Modifiers Program, National

Cancer Institute, NIH, Frederick, MD). This GM-CSF (manu-

factured by Immunex Corp., Seattle, WA) had a specific activity

of 1.4 x 106 IU/�i.g. Lyophilized GM-CSF was reconstituted

with sterile water, diluted to 100 p�g/ml with XVIVO- 1 5, and

stored at - 80#{176}Cbefore use.

Recombinant human TNF-a was a gift from Dr. Douglas

Fraker (Department of Surgery, University of Pennsylvania,

Philadelphia, PA). This TNF-a (manufactured by Knoll AG,

Ludwigshafen, Germany) had a specific activity of 8.2 X 106

units/mg protein as measured in the L929 cytotoxicity assay

without adding actinomycin D. Sterile, lyophilized TNF-a (0.79

mg/vial) was first reconstituted with 1 ml of sterile water before

further dilutions in culture medium.

Leukapheresis Collections. All leukapheresis collec-

tions from G-CSF-primed normal donors and cancer patients

were obtained through appropriate arrangements and Institu-

tional Review Board for Medicine-approved consent forms and

protocols established at the University of Michigan Medical

Center.

CD34� HSC Enrichment/Depletion. Normal donors

serving as the source of HSCs for allogeneic transplants re-

ceived 8 p.g/kg recombinant human G-CSF twice daily for 4

consecutive days before collecting the mobilized PBMCs by

leukapheresis. To obtain mobilized PBMCs from cancer (NHL

and multiple myeloma) patients, 4 g/rn2 cyclophosphamide (Cy-

toxan; Bristol-Myers Squibb, Princeton, NJ) followed by 10

��.g/kg recombinant human G-CSF per day were administered

until completion of the leukaphereses.

CD34� HSCs were isolated from mobilized PBMCs from

normal donors and cancer patients using either the Isolex-SO

(research scale) or Isolex-300 i (clinical scale) magnetic cell

separation system (Baxter Immunotherapy, Irvine, CA) as de-

scribed previously (24). The separations were performed on

whole, unmanipulated leukapheresis collections according to the

manufacturer’ s standard operating procedures. The purity of

CD34� HSCs in the positive fraction as well as the level of

CD34� HSC contamination in the negative fraction was deter-

mined by FACS analysis (see below).

Generation of DCs. For clinical applications, we have

refined a method to generate DCs from human PBMCs that uses

serum-free medium (25). The cells in the negative fraction from

the CD34� cell selection device or non-CD34� cell-depleted

(i.e. , unfractionated) mobilized PBMCs were washed twice in

HBSS (Life Technologies, Inc., Gaithersburg, MD) and resus-

pended in XVIVO-l5 medium at a concentration of 1.3 X 106

cells/mi. Three ml of this cell suspension were plated in each

well of 6-well tissue culture plates (Costar Corp., Cambridge,

MA) and incubated at 37#{176}Cin 5% CO, for 2 h. The nonadherent

cells were gently removed by pipetting and discarded, and 3

mI/well XVIVO-l5 medium containing GM-CSF (100 ng/ml)

and IL-4 (50 ng/mi) were added. All cultures were maintained at

37#{176}Cin 5% CO2 for 7 days. At day 7, culture medium was



Clinical Cancer Research 2711

exchanged with fresh XVIVO-15 medium containing GM-CSF/

IL-4 with TNF-a (10 ng/ml). The cultures were maintained for

another 7 days at 37#{176}Cin 5% CO2. We have found that 14-day

DCs treated with TNF-a at day 7 exhibited the characteristic

cellular projections and veils, which showed continuous exten-

sion, retraction, and reorientation (25).

At day 14, the adherent cells (DCs) were loosened using a

sterile cell scraper. Gentle scraping was necessary because DCs

cultured in serum-free XVIVO-l5 (as opposed to DCs cultured

in human AB- or FCS-containing RPMI 1640) became flattened

and adherent to the plastic tissue culture plates. The medium and

the loosened cells were transferred to sterile 50-ml conical

polypropylene tubes (Corning, Inc., Corning, NY). The DCs

were washed in XVIVO-l5 medium and counted before further

phenotypic and functional analysis (26). DC yields were calcu-

lated based on cell size (large) and morphological appearance

(veiled), with a high surface coexpression of HLA-DR and

CD83/CD86 but not CD14.

Isolation of T Cells from Mobilized PBMCs. Human T

cells were purified from mobilized PBMCs (i.e., post-Ficoll)

using nylon wool columns as described previously (27). Briefly,

20-ml syringe barrels containing 1.6-2.0 g of scrubbed nylon

wool (for a maximum loading capacity of 3 X 108 cells) were

sterilized by autoclaving and prepped with warm (37#{176}C)CM.

Mobilized PBMCs were added at �7.5 X i0� cells/ml CM, and

the columns were incubated in an upright position in a 37#{176}C,5%

CO2 humidified incubator for 45 mm. The nonadherent cells

(i.e., T cells) were collected as described previously (27). The

harvested T cells were centrifuged for 10 mm at 1500 rpm and

counted. The T cells (1 X 108 cells/mI/vial) were then cryopre-

served and stored in liquid nitrogen vapor phase until use. The

purity of the T cells was >90% by FACS phenotypic analysis of

the CD3 surface expression.

Allogeneic MLR Assay. Nylon wool-purified T cells

(100,000) from mobilized PBMCs of adult normal donors or

cancer patients were cultured in 96-well U-bottomed micro-

plates (Costar Corp.) with differing numbers of DCs. Plates

were incubated at 37#{176}Cin 5% CO2. Cellular proliferation was

measured on day S after an 18-24-h pulse with [3H}dThd at 1

�i.Ci/well (6.7 Ci/mmol; DuPont New England Nuclear, Boston,

MA) by a liquid scintillation counter ( 1205 Betaplate; Wallac,

Inc., Gaithersburg, MD). In some experiments, the level of

T-cell proliferation induced by PHA (Sigma Chemical Co., St.

Louis, MO) was included as a positive control to determine the

maximum level of proliferation induced by optimal stimulation

by this known lectin. PHA (0.5 rig/well) was added to additional

triplicate wells of 1 X l0� T cells alone on day 0 or day 3 of

culture.

Antigen Presentation Assay. To measure the efficiency

of DC presentation of soluble antigen, autologous nylon wool-

purified T cells were cultured at 1 X i05 cells/well with varying

numbers of DCs pulsed with iT (1:100 dilution of the cryopre-

served:thawed stock) in 200 p.1 of XVIVO-15 medium in 96-

well U-bottomed microplates. Cultures were pulsed with 1 p.Ci/

well [3H]dThd (6.7 Ci/mmol) on day 4. Cellular proliferation

was measured by [3H]dmd incorporation 18-24 h later with a

liquid scintillation counter (Wallac).

FACS Analysis. Cell surface staining used direct immu-

nofluorescence (FACScan; Becton Dickinson, Mountain View,

CA), and the samples were analyzed using Cell Quest software

(Becton Dickinson). Staining was performed with the following

FITC- or PE-labeled mAbs: (a) CD34; (b) CDla; (c) CD1 lc; (d)

CD14; (e) CD8O; (J) CD86; (g) HLA-DR (from Becton Dick-

inson or PharMingen, San Diego, CA); and (h) CD83 (from

Coulterflmmunotech, Miami, FL). Antibodies were directed to-

ward the panel of cell surface markers, and the level of single-

color or two-color staining was compared with that of the

appropriate isotype-matched controls.

RESULTSDCs Can Be Generated from CD34� Cell-depleted Mo-

bilized PBMCs as Measured by Phenotype and Function.

To obtain DCs in adequate numbers for immunization purposes

from the same leukapheresis collection used for PBSCT, we

focused attention on the negative fraction of positively selected

CD34� HSCs after immunomagnetic bead column separation.

In 13 consecutive CD34� cell selection runs using leukaphere-

sis collections from G-CSF-primed normal donors as well as

NHL and multiple myeloma patients with a preselection CD34�

content ranging from 0.53-4.28%, consistently high enrich-

ments of HSCs were obtained in the positive fraction with their

substantial depletion from the negative fraction. Although the

purity of CD34� HSCs tended to be greater in mobilized

PBMCs of cancer patients compared to those of normal donors

(87.0 versus 96.5%, respectively), the levels of depletion from

the negative fraction were nevertheless substantial, with residual

contamination below possible detection by FACS instrumenta-

tion (range, 0.00-0.15%; data not shown).

In some cases, CD34� HSCs obtained from the clinical-

scale Isolex-300 i cell selection device were used for autologous

transplantation in patients (administered at 2 X 106 CD34�

cells/kg). These included CD34� HSCs from the mobilized

PBMCs of two NHL patients and one multiple myeloma patient.

These transplants were successful, with the recipients achieving

myeloid and platelet reconstitution within standard, defined time

frames (data not shown).

We next examined the capacity of the fraction of mobilized

PBMCs depleted of CD34� HSCs to generate DCs as defined

by phenotype and function. The DCs generated in this culture

displayed morphological and phenotypic characteristics of ma-

ture DCs. As shown by the representative FACS profile in Fig.

1 , DC cultures generated under these defined conditions from

the negative fraction of mobilized PBMCs depleted of CD34�

HSCs were positive for CD1a, CD1 lc, CD8O, CD86, and

HLA-DR cell surface markers but expressed low to negligible

levels of CD14. Table 1 shows the results of a series of suc-

cessful attempts to generate DCs from CD34� cell-depleted

mobilized PBMCs of both normal donors and cancer patients.

DCs could be generated from either mobilized PBMC source

with little, if any, difference in overall yield (i.e., 5.8 ± 5.4

versus 4. 1 ± 1 .4%) between G-CSF-primed normal donors and

cancer patients, respectively.

We next tested DC function in both allogeneic MLR and

soluble antigen presentation assays. In Fig. 2, DCs generated from

CD34� cell-depleted mobilized PBMCs ofboth a NHL patient and

a normal donor in the presence of GM-CSF, IL-4, and TNF-a were

tested for their capacity to stimulate a primary allogeneic MLR.



Fluorescence Intensity


‘-4

E

z

c)

Fig. I FACS phenotypic profile of DCs generated from the CD34�HSC-depleted negative fraction of separated mobilized PBMCs. TheseDCs typically expressed the costimulatory molecules CD8O and CD86as well as HLA-DR but did not express CDI4. In addition, CDla,

CD1 lc, CD13, CD16, CD32, and CD33 markers were expressed atvarying levels. DCs were analyzed after a 14-day culture of the CD34�

HSC-depleted mobilized PBMCs in human recombinant GM-CSF, IL-4,

and TNF-a as described in “Materials and Methods.” Staining was

detected by a panel of antihuman FITC- or PE-labeled antibodies.

Positively stained cells are displayed by the open histograms compared

to isotype-matched control mAbs (shaded histograms). The X axis is a

logarithmic scale of fluorescence intensity, and the Y axis representscounts.

Different numbers of DCs were cultured with a fixed number of

allogeneic T cells from different donors to achieve DC stimulator:

T-cell responder ratios of 1 : 10 and I :20. These ratios were found to

be optimal for inducing T-cell alloproliferative responses in our

previous studies (data not shown). As shown by the separate

representative experiments, DCs displayed potent capacity to stim-

ulate primary allogeneic MLRs in vitro. We also evaluated the

capacity of DCs generated from the CD34� cell-depleted fraction

of the mobilized PBMCs to stimulate an autologous T-cell response

to the soluble, defined antigen iT. Fig. 3 shows a representative

experiment of potent and specific anti-TI’ T-cell proliferative re-

activity to antigen-pulsed autologous DCs from a multiple my-

eloma patient.

DCs Generated from CD34� Cell-depleted Mobilized

PBMCs Do Not Differ from DCs Generated from Non-

CD34� Cell-depleted Mobilized PBMCs by Phenotype orAllostimulatory Capacity. Next we directly compared DCs

generated from unmanipulated mobilized PBMCs versus those

Table 1 Generation of DCs from CD34� cell-depleted mobilized

PBMCs

PBMC source Donor DC yield (%)“

Normal JB 18.72

JW 5.00LD 4.62OJ 6.05DS 5.07CB 2.22

Mean ± SD 5.78 ± 5.44Lymphomab PE’� 3.08

Ki 6.92WT’ 4.70WTC 3.42

WT� 3.70

Myelorna JRC 2.88

Mean ± SD 4.06 ± 1.39

a The DC yield was determined morphologically by typical veiled

appearance; by FACS analysis, these cells also coexpressed high levels

of the costimulatory molecules CD8O and CD86 as well as HLA-DR and

CD83, but they did not express CD14.a Intermediate-grade NHL patients.

C Mobilized PBMCs from these patients were selected for CD34�

cells using the clinical scale Isolex-300 i; all others were selected with

the research scale Isolex-50.

generated from the CD34� cell-depleted negative fraction of

mobilized PBMCs phenotypically and functionally. Fig. 4

shows the FACS analysis conducted on DCs from the mobilized

PBMCs of a NHL patient. Comparable levels of cell surface

expression were noted for CD1a, CD86, CD1 ic, CD8O, and

HLA-DR (and little, if any, detectable CD14 expression) be-

tween DC cultures generated from CD34� cell-depleted

PBMCs versus those from nondepleted mobilized PBMCs. High

levels of CD83 expression were also present on both sources of

DCs, indicating a mature stage (data not shown; Refs. 25 and

26). The capacity of these two sources of DCs to stimulate a

primary allogeneic MLR was also compared. As shown in Fig.

5, comparable levels of DC allostimulatory activity were ob-

tamed, regardless of whether the DCs were generated from

CD34� cell-depleted versus nondepleted mobilized PBMCs. Of

note, the level of allostimulation of responding T cells to DCs

(at a 10: 1 ratio) was nearly equivalent to the maximum prolif-

eration of T cells obtainable by PHA stimulation. When com-

parisons of DC yields were made, the CD34� cell-depleted

fraction tended to generate slightly greater numbers of DCs

compared to similar aliquots of unmanipulated mobilized

PBMCs from both normal donors and cancer patients (data not

shown).

DISCUSSION

We and others have shown that DCs pulsed with tumor-

associated antigen(s) in the form of whole cell lysates (4-7),

peptides (8, 14), proteins (28), RNA (29), or DNA (30) could

initiate primary MHC class I- or 11-restricted T-cell responses

that resulted in antitumor effects both in vitro and in vivo. Based

on these studies, attention has focused on the use of DCs to

enhance the host immune response to tumor-associated anti-

gen(s) in clinical vaccine strategies in humans with cancer (14,

31). We have recently initiated Phase I clinical trials of autol-



CL)

2(50)

15000

6(0)

Cl� 14000C

� (20004-Cl0. 10000

�8000C

6000

4000

2000DC alone 1:10 1:20 T.CeIIsalone

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

0


Intermediate Grade Lymphom

-±-

-z-

m � �DCalone 1:10 1:20 1-Cells alone

Dendritic Cell I T-Cell Ratios

Fig. 2 DCs generated from the CD34� HSC-depleted negative fractionof separated mobilized PBMCs are stimulatory in a primary 5-dayallogeneic MLR. Mobilized PBMCs from a normal donor (top panel)

and a NHL patient (bottom panel) were used to generate DCs: twoseparate mobilized PBMC donors served as the source of responder i

cells in the assay, as described in “Materials and Methods.” Stimulator

(DC):responder (i cell) ratios were I : 10 and 1:20. DCs were generatedin GM-CSF-, IL-4-, and TNF-a-supplemented cultures and tested at 14

days. The SD of replicate wells is shown by the error bars.

ogous tumor lysate-pulsed DCs as a vaccine in adult and pedi-

atric patients with advanced solid tumors. Efforts to expand this

strategy to the setting of PBSCT are now underway. Thus, the

identification of approaches that would generate large numbers

of DCs with potent antigen-presenting capacity, particularly in

the setting of PBSCT, would be an important step in the further

refinement of such a vaccine strategy.

In the current study, we focused attention on strategies

to obtain functional DCs from the same mobilized PBMCs

leukapheresis collection used to obtain highly selected

CD34� HSCs for transplantation. We have found that both

sources of CD34� cell-depleted and nondepleted mobilized

PBMCs could generate DCs of similar phenotype and func-

tion when cultured in the recombinant human cytokines GM-

CSF, IL-4, and TNF-a. Importantly, functional DCs could be

readily generated from the cryopreserved PBMC sources

upon thawing, which allows considerable flexibility in the

timing and repetitiveness of DC-based vaccine immuniza-

tions after CD34� HSC transplantation. DCs from both mo-

bilized PBMC sources displayed the typical phenotype of

high coexpression of the costimulatory molecules CD8O and

+

-I-

- - - EI�III�DCalone 1:10 :20 1-Cells alone

Dendritic Cell / T.Cell Ratios

Fig. 3 DCs generated from the CD34� HSC-depleted negative fractionof separated mobilized PBMCs from a multiple myeloma patient pos-

sess a potent antigen-presenting function. The proliferative response of

purified autologous CD4� T cells to ‘FT antigen presented by autologous

DCs was measured at day 5 as described in “Materials and Methods.”Stimulator (DC):responder (T cell) ratios were 1: 10 and 1:20. DCs weregenerated in GM-CSF-. IL-4-, and TNF-a-supplemented cultures and

tested at 14 days. The SD of replicate wells is shown by the error bars.

CD86 as well as CD83 and HLA-DR, with little, if any,

expression of CD14. In addition, these DCs, unlike CDI4’

monocytes/macrophages, were capable of potent primary

stimulation of allogeneic T cells and presentation of the

soluble antigen TT to autologous T cells in vitro, which are

hallmark functions of this cell type. Overall, our study con-

firms that of Tarte et a!. (32) and also extends their results by

now including intermediate-grade NHL patients in the anal-

ysis.

Prior studies had identified proliferating progenitors within

the CD34� cell fraction of human BM, cord blood, and adult

PBMCs that could be driven with cytokines, particularly GM-

CSF and IL-4 with or without TNF-a, to develop into potent

DCs over a 1-2-week period in culture (1, 3, 12, 13, 31). In

addition, DCs have been derived from precursors in unfraction-

ated PBMCs as well as from CDl4� blood monocytes. How-

ever, the vast majority of functional DCs generated from non-

mobilized PBMCs have been shown to be derived from both

CDl4� and CD2� precursors ratherthan from CD34� HSCs (1,

12, 26, 33). Thus, the demonstration that functional DCs can be

generated from the CD34� cell-depleted mobilized PBMCs

adds confidence that these DCs are those that should be capable

of potent APC activity in vito. These DCs could now be used in

vaccine strategies in the setting of CD34� HSC transplants in

cancer patients.

In the transplant setting, DCs could be pulsed with

relevant idiotype proteins or whole tumor lysates that serve

as tumor-associated antigens in B-cell lymphoma and multi-

pie myeloma as well as potentially in leukemia patients who

have relapsed after allogeneic transplant. In addition, several

known tumor-associated peptides recognized by cytolytic T

cells have been molecularly cloned and shown to be shared

(or common) for certain HLA haplotypes in breast/ovarian

cancer (namely HER2/neu and carcinoembryonic antigen;



CD34+ Cell Depleted CD34+ Cell non-Depleted

-

Cl

C

C

4.

CCC

CD8O

HLA-DR

� CD1a

E

z.� CD86

LI

CD11c

CD14


�& �

�

A�.

Fluorescence Intensity

Fig. 4 Comparison of FACS phenotypic profiles of DCs generated

from CD34� HSC-depleted versus CD34� HSC nondepleted (unma-nipulated) mobilized PBMCs from a NHL patient. DCs from both

sources expressed comparable levels of the costimulatory molecules

CD8O and CD86 as well as HLA-DR but did not express CD14. In

addition, these DCs also displayed comparable levels of a series of

CDI6. CD32, CD33, CDIa, CDI3, and CD1 Ic markers, with theexception of CD14. DCs were analyzed after a 14-day culture of therespective mobilized PBMCs in human recombinant GM-CSF, IL-4,

and TNF-a as described in “Materials and Methods.” Staining was

detected by a panel of antihuman FITC- or PE-labeled antibodies.

Positively stained cells are displayed by the open histograms compared

to the isotype-matched control mAbs (shaded histograms).

Refs. 34 and 35), which would offer the possibility of ex-

panding this DC vaccine approach to solid tumors (e.g.,

breast and ovarian tumors) as well. In the allogeneic (MHC-

matched, unrelated) transplant setting, the source of DCs

would be autologous to the CD34� HSC transplant and the

resulting progeny hematolymphoid lineages upon reconstitu-

tion. The immunization of patients with peptide/protein-

pulsed DCs in the allogeneic transplant setting offers the

intriguing possibility of augmenting graft versus tumor ef-

fects without augmenting the graft versus host reaction in the

transplant recipient. It is also conceivable, however, that the

high potency of DCs in antigen-presenting function could

uncover or enhance the reactivity to minor histocompatibility

antigens, which could, in turn, increase graft-versus-host

disease.

Aside from the fact that immunomagnetic bead separa-

tion is a rapid and effective approach for isolating engrafting

doses of CD34� HSCs from mobilized PBMCs, as shown in

1:160 1:320 � 1:640alone alone + P1-IA

Dendritic Cell / T-Cell Ratios

Fig. 5 DCs generated from both CD34C HSC-depleted and CD34HSC nondepleted (unmanipulated) mobilized PBMCs from a NHLpatient are comparable in their stimulatory activity in a primary, 5-day

allogeneic MLR at varying stimulator (DC):responder (T cell) ratios.

For comparison, the maximum T-cell proliferation induced by the lectin

PHA was used as the positive control, as described in “Materials and

Methods.” The SD of replicate wells is shown by the error bars.

the current study as well as in those of others (24, 36), we

demonstrated that ample numbers of DCs can also be gener-

ated from the same leukapheresis collection by using the

CD34� cell-depleted negative fraction of mobilized PBMCs.

Importantly, culture of this negative fraction in recombinant

cytokines GM-CSF, IL-4, and TNF-a resulted in overall DC

yields of 4-5% at 14 days. In terms of theoretical clinical

scale-up, this amount would represent 4-5 X 108 DCs for

every I X lO’#{176} mobilized PBMCs plated in culture after

CD34� HSC removal. This yield should be considered sub-

stantial, given the fact that as few as a median of 5 X 106

tumor antigen-charged DCs freshly obtained from whole,

unfractionated PBMCs have been shown in an early clinical

vaccine trial in follicular B-cell lymphoma patients (not

undergoing PBSCT or BMT) to augment cellular antitumor

reactivity in 4 of 4 immunized patients and cause complete or

partial tumor regressions in 3 of 4 of those patients (37). In

a recent reported clinical study in advanced melanoma pa-

tients (in the nontransplant setting) immunization with 1 X

106 autologous DCs/injection has resulted in some partial and

complete responses as well as in the induction of tumor-

specific cytotoxic T cells (38).

Collectively, our data demonstrate the feasibility of gener-

ating potent DCs from the same leukapheresis as isolated

CD34� HSCs used for transplant purposes. The advantages of

now attempting DC-based immunizations in the setting of

PBSCT include lower tumor burden, reduction (or elimination)

of tumor-induced immunosuppression, and the possibility of

biasing or educating the developing immune T-cell repertoire to

selectively target and potentially eliminate residual malignant

disease in the transplanted patient.

ACKNOWLEDGMENTSWe thank Dr. Douglas Fraker (University of Pennsylvania, Phila-

delphia, PA) for the provision of recombinant TNF-a and Dr. Satwant




Narula, Dr. Mary Ellen Rybak, and Chris DeLuca (Schering-Plough

Research Institute, Kenilworth, NJ) for the generous supplies of recom-

binant human GM-CSF and IL-4. We also thank Dr. Larry Baker of the

University of Michigan Comprehensive Cancer Center (Ann Arbor, MI)

and Dr. Paul Watkins, Dr. Blake Roessler, and Dorene Markel of the

University of Michigan General Clinical Research Center (Ann Arbor,

MI) for their efforts in establishing a dedicated leukapheresis and HSC

separation facility for clinical research studies. The provision of

Isolex-50 and Isolex-300 i devices by Dr. Dennis Van Epps and Baxter

Healthcare Corp. is greatly appreciated.

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1998;4:2709-2716. Clin Cancer Res D Choi, M Perrin, S Hoffmann, et al. peripheral blood can serve as a source of potent dendritic cells.stem cell transplantation: CD34+ cell-depleted mobilized Dendritic cell-based vaccines in the setting of peripheral blood

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