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

capacity of DC to capture and process antigens, transport them tolymph nodes, and therein activate naive T cells nominates them to a“specializedsubset of antigen-presenting cells,―as suggested bySteinman (3) and Knight and Stagg (10).

For cancer therapy, investigators have proposed using DC pulsed

with tumor antigen as therapeutic vaccines in vivo, as well as primingcancer antigen-specific T cells in vitro for use in adoptive T celltherapy (3—5,10, 13). However, isolation of adequate numbers hasbeen difficult, because DC are present in the peripheral blood in verylow numbers (3). Caux et a!. (14) showed that DC could be derivedand expanded from CD34@ HPC in umbilical cord blood by inducingdendritic cell differentiation and proliferation with GM-CSF plusTNF-a (14). The current experiments asked whether DC could besimilarly derived and expanded from CD34@ HPC in bone marrow orperipheral blood of cancer patients. CD34@ HPC are present in theperipheral blood in only low numbers, comprising less than 1% of theWBCs. However, the enrichment and isolation of CD34@stem cellsis now a standard procedure for stem cell support following high-dosechemotherapy. For our peripheral blood studies adequate numbers ofCD34@ cells could be positively selected by antibody affinity columns after stem cell mobilization by in vivo administration of G-CSFfor 3 to 4 days followed by leukapheresis.

The results demonstrated that culturing CD34@ HPC, which werederived either from BM or PBSC collections, with GM-CSF plusTNF-a induced differentiation of heterogeneous cell populations including cells expressing dendritic cell morphology and phenotype.The cultures containing DC were able to stimulate proliferation ofallogeneic T cells in vitro, the most common assay used to evaluatedendritic cell function. Critical to the development of a vaccinestrategy, the cultured DC should also function as APC for the stimulation of T cell responses to protein and peptides. Autologous T cellproliferation could be induced by loading DC with whole tetanustoxoid protein as well as with a peptide derived from the normal

amino acid sequence of HER-2/neu. This oncogenic protein is overexpressed in adenocarcinomas of different origins, such as breastcancer, and is a potential target for immunotherapy (15—18).Thedemonstrated ability to procure increased numbers of DC might allowthe development of dendritic cell-based vaccines and T cell therapyregimens.

ABSTRACT

Dendritic antigen-presenting cells are considered to be the most effecdye stimulators of T cell immunity. The use of dendritic cells has beenproposed to generate therapeutic T cell responses to tumor antigens incancer patients. One limitation Is that the number of dendritic cells inperipheral blood Is exceedingly low. Dendritic cells originate from CD34@hematopoletic progenitor cells (HPC) which are present in the bonemarrow and in small numbers in peripheral blood. CD34@ HPC can bemobilized Into the peripheral blood by in vivo administration of granulecyte-colony-stimulating factor. The aim of the current study was to determine whether functional dendritic cells could be elicited and grown invitro from CD34@HPC derived from bone marrow or granulocyte-colonystimulaflng factor-mobilized peripheral blood. Culture of CD34@ HPCwith granulocyte-macrophage-colony-stlmulating factor and tumor necro515 factor a yielded a heterogeneous cell population containing cells with

typical dendritic morphology. Phenotypic studies demonstrated a loss ofthe CD34 molecule over 1 week and an increase in cells expressing surfacemarkers associated with dendritic cells, CD1a, CDSO (B7IBB1), CD4,CD14, HLA-DR, and CD64 (FciRI). Function was validated in experimeats showing that CUltUredcells could stimulate proliferation of allogeneic CD4' and CD8@T lymphocytes. Antigen-presenting capacity wasfurther confirmed in experiments showing that cultured cells could effectively stimulate tetanus toxoid-specific responses and HER-2/neu peptidespecific responses. The derivation and expansion of dendritic cells fromcultured bone marrow or granulocyte-colony-stimulating factor-mobilized 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 responsesin vivo including sensitization of MHC-restricted T cells and development of T cell-dependent antibodies (1—7).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, DCtake up antigen, migrate through the afferent lymphatic system to thelymphoid organs, and efficiently present antigen to T cells (3). DC arethought to be the major APC type involved in triggering primary Tcell responses (10). A number of studies have demonstrated thathuman DC derived from the peripheral blood are more potent APC

than peripheral blood-derived monocytes or B cells (11, 12). The

Received 9/6/94; accepted 1/3/95.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges.Thisarticlemustthereforebe herebymarkedadvertisementin accordancewith18 U.S.C. Section 1734 solely to indicate this fact.

1 ‘Pals work has been supported by Deutsche Forschungsgemeinschaft Grant

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

2 Berlex Oncology Foundation Fellow. 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 cells; HPC,hematopoietic progenitor cells; PBSC, peripheral blood stem cells; BM, bone marrow;G-ChF, granulocyte-colony-stimulatingfactor;GM-CSF,granulocyte-macrophage-colony-stimulating factor; FACS, fluorescence-activated cell sorting; TNF-a, tumor necrosisfactor a; MLR, mixed leukocyte reaction; PE, phycoerythrin.

MATERIALS AND METHODS

Source of Cells. Peripheralbloodmononuclearcells were obtainedfrombreast cancer patients as well as from healthy donors, bone marrow samplesfromhealthydonorsonly.PBSCwerecollectedfrompatientsaboutto undergoautologous stem cell transplantation for breast cancer. The majority of thepatients had advanced stage breast cancer in complete or partial remission withstable clinical condition at the time of collection. Patients were not activelyreceiving chemotherapy. Briefly, patients were administered G-CSF at a doseof 10 pg/kg daily for 3 to 4 days to mobilize stem cells. Leukapheresis wasperformed on day 3 or 4. Aliquots (3 ml) of the leukapheresis product wereused to purify CD34@HPC.

1099

Generation of Immunostimulatory Dendritic Cells from Human CD34+

Hematopoietic Progenitor Cells of the Bone Marrow andPeripheral Blood'

Helga Bernhard, Mary L. Disis,2 Shelly Heimfeld, Susan Hand, Julie R. Gralow, and Martin A. CheeverDepartment 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.]

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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 peripheralblood. CD4@ and CD8@ T lymphocytes were purified from peripheral blood

mononuclear cells or PBSC using the cell separation system Ceprate LC Kit

(CelIPro, Bothell, WA; Ref. 19). First, samples were processed using FicollHypaque density gradient centrifugation (Pharmacia, Piscataway, NJ). Cellsobtained from the interface were washed and resuspended in PBS-1% BSA. Inorder to purify CD34@ HPC, up to 108 cells/mI were incubated for 25 mm onice with 40 @g'mlbiotinylated mAb anti-CD34 in PBS-1% BSA. Purificationof 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. Cellswere washed with PBS-l% BSA and incubated with a biotinylated antimousemAb for an additional 25 mm. The biotinylated cells were washed withPBS-1% BSA to remove any unbound antibody. This fraction, in a volume of1 ml PBS-5% BSA/lO' cells, was applied to the Ceprate column, whichcontained sterile avidin-coated polyacrylamide beads. After washing with

PBS, the attached cells were removed from the beads by mechanical agitationand eluted with PBS.

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

by staining with anti-CD4 and anti-CD8 mAb, respectively. The selected Icells were not activated through the selection procedure. Purity of CD344 cellsdepended on the source of the HPC. CD34@ cells isolated from bone marrow

regularly showed a purity of 80% to >95%, whereas the purity from PBSCderived CD34@ cells ranged from 30 to 80% depending on the response toG-CSF treatment of the individual patient.

Dendritic Cell Culture Derived from HPC. DC were generatedusing amodified method, described by Caux et al. (14), for generating DC fromumbilical cord blood. All cell cultures were initiated from CD34@ HPC, whichhad been isolated through positive selection by immunoaffinity columns. Thepurified 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 culturemedium was further supplemented with GM-CSF (100 ng/ml) (ScheringPlough, 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 4—5days. After12—16days of culture time, cells were harvested and used for phenotyping andfunctional assays.

Monocyte Culture. Monocytes were enriched as described previously (2).In short, PBSC (2 x l06/ml) were cultured in supplemented RPMI 1640 inPetri dishes (100 mm; Falcon, Lincoln Park, NJ). After 36 h of culture at 37°C,

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 hwere used as an enriched source of monocytes. In FACS analysis, 70% of the

adherent cells displayed the monocyte marker CD14.

Flow Cytometric Analysis. Ihe following mAbs were used for FACSanalyses: 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, antiCD4, anti-CD8, anti-CD56, anti-CD19 (Becton Dickinson, San Jose, CA);anti-HLA-DR-PE (Olympus Corp., Lake Success, NY), FIIC-F(ab')2 fragment goat anti-mouse IgG (Zymed, San Francisco, CA).

Double-color fluorescence staining of cell cultures was carried out bysequential incubation of mAbs. Briefly, cells were resuspended in PBS-1%BSA and seeded into microtiter plates at a final concentration of iO@cells/well.In order to reduce nonspecific binding, 10 @lgoat immunoglobulin (3 mg/mI)were added to each well. After 10 mm, cells were incubated with the unconjugated mAbs at saturating concentrations for 10 mm on ice, washed twice

with 150 @.dPBS-l% BSA and stained with FIIC-F(ab')2 fragment conjugatedgoat anti-mouse IgG for an additional 20 mm. Following two washes withPBS-1% BSA, 10 pi mouse immunoglobulinswere added for 10 mm to reducecross-reactivity. Finally, cells were incubated with PE-labeled mAb for 20 mm,washed twice with PBS-1% BSA and fixed with 1% paraformaldehyde.Negative controls were performed with FITC-F(ab')2 goat anti-mouse IgG anda PE-conjugated unrelated murine mAb (Becton Dickinson). Fluorescenceanalyses were performed with a FACScan flow cytometer (Coulter).

MLR. Naive CD4@or CD8@I lymphocytes (5 X 104/well) isolatedthrough the Ceprate LC Kit from normal donor peripheral blood lymphocytes

were used as responder cells. Dendritic cell cultures were harvested after12—16days, irradiated with 30 Gy, and added to the responder cells at differentconcentrations (102-104/well). The assay was performed in 96-well round

bottomed plates (Corning, Coming, NY) at 37°C.The medium used consistedof equal parts of EHAA (Biofluids) and RPMI 1640 (Gibco) with 10 mML-glutamine, 200 units/ml penicillin, 200 units/ml streptomycin, 2.5 X iO@ M2-mercaptoethanol, and 10% human serum (human AR CELLect; ICN Flow,Costa Mesa, CA). After 4 days, cells were pulsed with 50 pJ/well [3H]thymidine (1 mCi/rn]) for 8 h and counted. Data are shown as the mean of fourreplicates.

Antigen-presenting Assay. For induction of antigen-specific T cell responses two different antigens were used. Peptide p42—56,derived from theamino acid sequence of HER-2/neu protein, is 15 amino acids in length andwas chosen based on an increased probability of binding to Class II MHCmolecules (17). The peptide was synthesized and purified by Dr. P. S. H. Chou(University of Washington, Seattle, WA). Preservative-free tetanus toxoid waschosen as a representative protein antigen (Wyeth).

Dendritic cell cultures were harvested, spun, and resuspended in equal

amounts of EHAA and RPMI 1640 supplemented as described above. Different cell concentrations were loaded with tetanus toxoid protein (25 p@g/ml)orHER-2/neu p42—56 peptide (50 @g/ml). Following an incubation period for 1

to 2 h at 37°C,the pulsed dendritic cell population was irradiated (30 Gy) andadded to autologous CD4@ I lymphocytes that acted as responder cells(5 X 104/well).Proliferativeresponsewas measuredas a functionof thymidineuptake and results are shown as the mean of four replicates.

RESULTS

DC Can Be Generated from CD34@ HPC from BM and Peripheral Blood in the Presence of GM-CSF and TNF-a. Initialexperiments asked whether the methods developed by Caux et a!. (14)for growing DC from cord blood could induce the growth of DC fromadult bone marrow. CD34@ HPC from BM were enriched by positiveselection (90% purity) and cultured with GM-CSF plus TNF-a for 12to 16 days. Results showed that the total cell number increasedapproximately 40-fold, ranging from 20- to 80-fold (Fig. 1A). Duringcell growth the CD34@ HPC population lost the CD34 marker whichwas undetectable by day 9, and cells with a more differentiatedmorphology appeared (Fig. 1B). In order to follow the differentiation

of CD34@ HPC into DC, the CD1a molecule, known as a marker fordendritic Langerhans cells, was used. CD1a expression graduallyincreased, reaching a maximum level on day 15 (20). At that timepoint the cultured population was heterogeneous, and the CD1a molecule was displayed by 30—60% of the cells, depending on theindividual cell culture. The relative number of CD1a expressing cells

200

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1 5 9 11 15 19 1 5

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Fig. 1. Kinetics (A) and phenotype (B) of dendritic cell generation from BM-derivcdCD34@HPC cultured with GM-CSF and TNF-co.Expansion of the CD34@HPC startingpopulation during a 19-day culture period is shown. CD34@HPC lost the CD34 markerduring 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 relatedto the expanding monocytes.

1100

Table 1Phenotype of HPC-derivedDCSurface

antigensHPC-derivedDCCD1al0-.60@'CD410-90CD1420-80CD8O10-60HLA-DR50-90CD34<5CD3<5CD8<5CD19<5CD56<5

GENERATION OF IMMUNOSTIMULATORYDENDRITIC CELLS

molecule (Fig. 4). All of the CD1a-positive cells were also positive forthe activation molecule CD8O (B7IBB1). The majority of CD1a@ cells(80—90%)were positive for CD4, which is known to be expressed bycultured DC (20). Subpopulations of CD1a@ cells (30—50%) werepositive for the monocyte marker CD14, which indicates that the

CD1a@ population can subdivide into CD1a@/CD14— cells andCD1a@/CD14@ cells.

Allogeneic T Lymphocyte Proliferation Can Be Induced by DCGenerated from CD34@ HPC. Cultured cell populations containingDC were tested for the capacity to induce proliferation of allogeneicT cells. After 12—14days of culture, cells generated from BM ormobilized peripheral blood HPC-derived CD34@ HPC were used as

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Fig. 2. Morphology of adherent cells generated from CD34@ HPC in response toGM-ChFand1'NF-a examinedunderphase-contrastmicroscopy.Adherentcells displaya dendritic morphology with delicate membrane projections. X 200.

B7/BB1

@/J \jdecreased after day 15 of culture due to expansion of cells withmonocyte and macrophage morphology. In the experiment presented,15 X 10@CD1a@cellswerederivedfrom a startingpopulationof2 X 10@CD34@ HPC.

Similar growth was observed for both BM- and peripheral bloodderived CD34@ HPC. The amount of cell growth strongly correlatedwith the degree of purity of the starting CD34@ cell population. Thisobservation was documented in 22 independent cell cultures. Periphera] blood-derived CD34@ HPC were, in general, less pure (30—80%)than BM-derived HPC (80 to >95%) after positive selection andexpanded to a lesser degree (5—20-fold)proportional to the percentageof CD34@ cells in the initial culture.

Cultured Cells Derived from CD34@ HPC Display Heterogeneous Morphology and Phenotypic Markers of DendriticlLangerhans Cells and Monocyte/Macrophages Including the Expressionof Class II MHC and Costimulator CD8O (B7IBB1) Molecules.The morphology of cultured cells was evaluated at days 12—14.During 2 weeks of culture the homogeneous population of smallmononuclear cells differentiated into a heterogeneous population ofadherent and nonadherent cells which could be distinguished byphase-contrast microscopy as three major cell populations. Approximately 50% of the total cells were adherent or loosely adherent, largeand vacuolated, with or without a few short membrane projections,typical features of monocytes and macrophages. The second majorcell population consisted of small, round, and nonadherent cellswithout vacuoles or projections which represented about 30% of thecell culture. This morphologically defined second cell populationcould not be categorized as a known functionally differentiated celltype. The third and smallest population (20%) contained mostlyadherent cells with a dendritic cell morphology with long delicatemembrane projections (Fig. 2). The cells with dendritic morphologywere generally overgrown by typical macrophages after 2 weeks.

Immunofluorescence analyses were performed between days 12and 15 with BM- and peripheral blood-derived dendritic cell cultures.

Representative FACS analyses are presented in Fig. 3. The culturedcells did not display cell surface markers for B lymphocytes (CD19),T cells (CD3, CD8), or natural killer cells (CDS6). Surface markersexpressed 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 ofthese markers varied from culture to culture, as shown in Table 1.Results were similar for populations generated from BM or peripheralbloodHPC.

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

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Fig. 3. Cell surface phenotype of BM- and peripheral blood-derived CD34@ cellscultured for 2 weeks in the presence of GM-CSF and TNF-a. Dashed lines, stainings withisotype-matched nonreactive control mAb; bold lines, staining with mAb as indicated. Thecell 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

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 levelsof expression of CD1a and CD8Owere routinely found in cultures of the highest initialCD34 purity.

b Percentage of positive cells.

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GENERA11ONOF IMMUNOS11MULATORYDENDRI11CCELLS

stimulator cells for allogeneic CD4@ as well as CD8@ T lymphocytesthat had been isolated using positive selection (>95% purity). Dendritic cell cultures derived from either BM (4/4) or PBSC (6/6)induced a strong proliferative T cell response for both CD4@ andCD8@ T lymphocytes, as demonstrated by an increased thymidineincorporation (Fig. 5). The number of T cells/well was held constantand the number of cultured DC as stimulator cells varied. The level ofT cell proliferation was dependent on the number of DC/well. The Tcells were not observed to be activated by the method of purificationused, as determined by a lack of thymidine incorporation withoutstimulator cells added.

DC Generated from CD344 HPC Can Present Antigens toAutologous CD4@ T Lymphocytes. The ability of positively selected CD4@ T cells from patients with breast cancer to respond toprotein and peptide antigens presented was examined in five individuals. Autologous CD4@ T lymphocytes purified from frozen PBSCacted as responder cells and PBSC-derived dendritic cell cultures asAPC. Soluble protein antigen or synthetic peptide was added tovariable numbers of cultured DC with a fixed number of autologousCD4@ cells (Fig. 6). Tetanus toxoid was used as a representative recallprotein. The peptide used, denoted as HER-2/neu p42—56,was a15-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 ofCD4@ T lymphocytes to tetanus toxoid and HER-2/neu peptide wasobserved to increase. In this patient, the response to tetanus toxoidwas greater than the response to HER-2/neu peptide. Of note, DCalone at higher numbers induced a T cell response presumably reflecting an autologous MLR. In a second patient, the capacity of DCgenerated from CD34@ HPC to present peptides was compared to

Fig. 5. MLR using BM-derived dendritic cell cultures as stimulator cells and aliogeneicCD4@and CD8@T lymphocytes as responder cells. T lymphocyte proliferation can beinduced 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 inducingalloreactivity.

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Fig. 6. Antigen presentation by dendritic cell cultures. Antigen-pulsed PBSC-dcrlvedDC from a breast cancer patient induced a protein- or peptide-specific CD4@response.HER-2/neu p42—56peptide, @,tetanus toxoid protein, t no antigen, A.

monocytes. As shown in Fig. 7, peptide-pulsed DC induced autologous CD4@ T cells to proliferate. In comparison, monocytes from thesame patient were not able to induce autologous CD4@ T cells toproliferate. Again, DC alone stimulated T cell proliferation. In three ofthe five patients tested, a proliferative response to HER-2/neu could

not be detected.

DISCUSSION

Developing the immunogenic potential of DC for cancer therapyrequires better access to this rare cell type (3). This report demonstrates that DC can be derived from positively selected CD34@ HPC.Culturing positively selected CD34@ HPC with TNF-a and GM-CSFresulted in an approximately 40-fold expansion of the starting cellnumber over 2 weeks and the generation of CD34-negative DC in anumber equivalent to at least the number of starting CD34@ cells.

CD34@ HPC derived from either G-CSF-mobiized peripheral bloodor BM are capable of acting as the starting population. The advantagesof using BM over PBSC are a greater purity of CD34@ stem cellsfollowing cell separation (80 to >95% versus 30—80%), and subsequent better cell growth (20—80-fold versus 5—20-fold). The collection of PBSC results in a higher total stem cell number afterleukapheresis. Since the purity of CD34@ stem cell preparationsvaried from donor and source, it cannot be formally ruled out that DCoriginated from CD34-negative DC progenitors. However, the stronger growth of highly pure CD34@ from the BM compared to lowpurity CD34 preparations from the peripheral blood indicates that DCcultures are derived mainly from CD34@ cells. This hypothesis issupported by single-cell experiments, which revealed that DC cobnies originate from CD34@ cells of the BM, as recently reported byReid et aL (21, 22).

The rationale for using TNF-cs in addition to GM-CSF was drawn1102

CD4

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Fig. 7. Comparison of dendritic cell cultures and monocytes, from a breast cancerpatient, as APC. DC and monocytes were loaded with HER-2/neu p42—56(R) or noantigen (Cl). Peptide-pulsed PBSC-derived DC induced a specific CD4@ response toHER-2/neu p42—56peptide, whereas monocytes from the same patient were not able topresent the peptide to the autologous @T@+T lymphocytes.

from the experience of others who showed that TNF-a modifiesGM-CSF-induced myelopoiesis and facilitates the development of DCfrom cord blood CD34@ HPC (14). Using this regimen to culturepositively selected CD34@ HPC from BM or peripheral blood resultedin a heterogeneous population of three major cell types based onmorphology: DC, monocytes, and small round cells of unknownfunction. The DC were mostly adherent and displayed a typicalmorphology with delicate membrane projections. The monocyteswere largely adherent and vacuolated and formed a tight cell networkwhich tended to overgrow the DC after 2 weeks. Both DC andmonocytes were expanded under the same conditions. It is possiblethat they may share the same precursor, a concept recently presentedby Reid et a!. (21, 22), and is supported by the observation that fullydifferentiated monocytes could be a source for generating veiledaccessory cells when cultured in serum-free medium (23, 24). Futureattempts at generating DC from CD34@ HPC should explore theinfluence of other cytokines to drive HPC differentiation into a morehomogeneous population of DC. Cytokines of interest include interleukin 1 which signals interleukin 1 receptor to up-regulate GM-CSFreceptors on epidermal DC; interleukin 4 which drives monocytes intocells with dendritic processes, down-regulates monocyte markers, andenhances antigen-presenting capacity; and interleukin 6 which stimulates monocyte-derived Langerhans cells via autocrine cytokineproduction (24—28).

The cell populations derived by culture of CD34@ HPC lost theCD34@ marker, were positive for markers known to be expressed byDC and monocytes (CD1a, CD4, CD8O, and Class II MHC; Refs. 3,14, 29—33),and lacked surface markers for B, T, and natural killercells (CD3, CD19, CD8, and CD56). The studies focused particularlyon the CD1a molecule which is associated with the immunostimulatory cell fraction from HPC-derived cell cultures (14). Double-colorimmunofluorescence showed that the majority of CD1a@ cells ex

GENERATION OF IMMUNOSTIMULATORY DENDRITIC CELLS

pressed CD4 and CD8O (B7/BB1). Approximately one half of theCD1a@ cells expressed CD14, a monocytic marker. It is not clearwhether only the CD1a@/CD14 cells should be designated as “dendritic cells―or whether some DC are CD1a@/CD14@. Others haveshown that DC can be sorted from dimly stained CD14@ cells (34).An important issue not addressed is possible functional differences inantigen 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@ Tlymphocytes (2, 3, 14, 35). Capacity to induce strong alloreactivitywas consistently shown using DC cultures derived from either bonemarrow (4/4) or mobilized peripheral blood HPC (6/6). In contrast,the ability to stimulate protein- and peptide-specific responses wasless consistent. In the experiments presented, cultured DC effectivelystimulated tetanus toxoid-specific responses and HER-2/neu peptidespecific responses (2/5). However, some dendritic cell cultures stimulated strong allogeneic responses, but not protein- and peptidespecific responses (3/5). The lack of a detectable antigen responsemight be due to a low precursor T cell frequency in those patients.Another reason for the preferential ability of DC to stimulate allogeneic responses might be a down-regulation of the ability to presentexogenous antigen after maturation in culture. It is known that intactprotein is presented best by immature DC (5, 27, 36, 37). A majorissue that remains is identifying culture conditions that expand DCwhich consistently retain the ability to process and present exogenousantigens. Other studies, as well as the data presented here, demonstrate augmentation of MLR with human DC derived from progenitorcells (14, 38). Results from our laboratory, however, show that cxtrapolation of dendritic cell function from MLR to specific antigenpresenting capability is not necessarily appropriate. Although functionin a MLR is reproducible, these same DC would have variablefunction when presenting specific antigens. Subsequent studies shouldfocus on the interrelationship between cell growth and the functionalability to stimulate protein- and peptide-specific responses and shouldutilize antigen-presenting function rather than cell number or allogeneic MLR for optimization of culture conditions. The availability ofsufficient numbers of efficient antigen-presenting DC should facilitatethe 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 KevinWhitham for manuscript preparation. In addition, we thank the members of theautologous bone marrow transplant team, leukapheresis unit, and cryopreservation unit at the Fred Hutchinson Cancer Research Center for supplying theperipheral blood stem cells used in these studies.

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REFERENCES

1. Steinman, R. M., and Cohn, Z. A. Identification of a novel cell type in peripherallymphoid organs of mice. J. Exp. Med., 137: 1142—1162,1973.

2. Young, J. W., and Steinman, R. M. Dendritic cells stimulate primary human cytolyticlymphocyte responses in the absence of CD4@ helper T cells. J. Exp. Med., 171:1315—1332,1990.

3. Steinman, R. M. The dendritic cell system and its role in immunogenicity. Annu. Rev.Immunol., 9: 271—296,1991.

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