Generation of Immunostimulatory Dendritic Cells from Human CD34+ Hematopoietic Progenitor Cells of the Bone Marrow and Peripheral Blood'

[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 capacity of DC to capture and process antigens, transport them to lymph nodes, and therein activate naive T cells nominates them to a Dendritic antigen-presenting cells are considered to be the most effec â€œspecializedsubset of antigen-presenting cells,â€•as suggested by dye stimulators of T cell immunity. The use of dendritic cells has been Steinman (3) and Knight and Stagg (10). proposed to generate therapeutic T cell responses to tumor antigens in cancer patients. One limitation Is that the number of dendritic cells in For cancer therapy, investigators have proposed using DC pulsed peripheral blood Is exceedingly low. Dendritic cells originate from CD34@ with tumor antigen as therapeutic vaccines in vivo, as well as priming hematopoletic progenitor cells (HPC) which are present in the bone cancer antigen-specific T cells in vitro for use in adoptive T cell marrow and in small numbers in peripheral blood. CD34@ HPC can be therapy (3â€”5,10, 13). However, isolation of adequate numbers has mobilized Into the peripheral blood by in vivo administration of granule been difficult, because DC are present in the peripheral blood in very cyte-colony-stimulating factor. The aim of the current study was to de low numbers (3). Caux et a!. (14) showed that DC could be derived termine whether functional dendritic cells could be elicited and grown in and expanded from CD34@ HPC in umbilical cord blood by inducing vitro from CD34@HPC derived from bone marrow or granulocyte-colony dendritic cell differentiation and proliferation with GM-CSF plus stimulaflng factor-mobilized peripheral blood. Culture of CD34@ HPC with granulocyte-macrophage-colony-stlmulating factor and tumor necro TNF-a (14). The current experiments asked whether DC could be 515 factor a yielded a heterogeneous cell population containing cells with similarly derived and expanded from CD34@ HPC in bone marrow or typical dendritic morphology. Phenotypic studies demonstrated a loss of peripheral blood of cancer patients. CD34@ HPC are present in the the CD34 molecule over 1 week and an increase in cells expressing surface peripheral blood in only low numbers, comprising less than 1% of the markers associated with dendritic cells, CD1a, CDSO (B7IBB1), CD4, WBCs. However, the enrichment and isolation of CD34@stem cells CD14, HLA-DR, and CD64 (FciRI). Function was validated in experi is now a standard procedure for stem cell support following high-dose meats showing that CUltUredcells could stimulate proliferation of alloge chemotherapy. For our peripheral blood studies adequate numbers of neic CD4' and CD8@T lymphocytes. Antigen-presenting capacity was CD34@ cells could be positively selected by antibody affinity col further confirmed in experiments showing that cultured cells could effec tively stimulate tetanus toxoid-specific responses and HER-2/neu peptide umns after stem cell mobilization by in vivo administration of G-CSF specific responses. The derivation and expansion of dendritic cells from for 3 to 4 days followed by leukapheresis. cultured bone marrow or granulocyte-colony-stimulating factor-mobi The results demonstrated that culturing CD34@ HPC, which were lized CD34+ HPC may provide adequate numbers for testing of dendritic derived either from BM or PBSC collections, with GM-CSF plus cells in clinical studies, such as vaccine and T cell therapy trials. 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 INTRODUCTION allogeneic T cells in vitro, the most common assay used to evaluate DC3 are APC that are critical for the initiation of T cell responses dendritic cell function. Critical to the development of a vaccine in vivo including sensitization of MHC-restricted T cells and devel strategy, the cultured DC should also function as APC for the stim opment of T cell-dependent antibodies (1â€”7).DC originate from ulation of T cell responses to protein and peptides. Autologous T cell @J@34+pluripotent HPC in the bone marrow and migrate as immature proliferation could be induced by loading DC with whole tetanus cells to nonlymphoid tissues such as skin (Langerhans cells), mucosa, toxoid protein as well as with a peptide derived from the normal and tumor (1, 8, 9). During antigen-induced immune responses, DC amino acid sequence of HER-2/neu. This oncogenic protein is over take up antigen, migrate through the afferent lymphatic system to the expressed in adenocarcinomas of different origins, such as breast lymphoid organs, and efficiently present antigen to T cells (3). DC are cancer, and is a potential target for immunotherapy (15â€”18).The thought to be the major APC type involved in triggering primary T demonstrated ability to procure increased numbers of DC might allow cell responses (10). A number of studies have demonstrated that the development of dendritic cell-based vaccines and T cell therapy human DC derived from the peripheral blood are more potent APC regimens. than peripheral blood-derived monocytes or B cells (11, 12). The

Received 9/6/94; accepted 1/3/95. MATERIALS AND METHODS Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage charges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith Source of Cells. Peripheralbloodmononuclearcellswere obtainedfrom 18 U.S.C. Section 1734 solely to indicate this fact. 1 â€˜Pals work has been supported by Deutsche Forschungsgemeinschaft Grant breast cancer patients as well as from healthy donors, bone marrow samples Be1579/11 (H. B.) and NIH Grants ROl CA57851 and 5 ROl CA49850 (S. H., J. R. G., fromhealthydonorsonly.PBSCwerecollectedfrompatientsaboutto undergo M. A. C.). autologous stem cell transplantation for breast cancer. The majority of the 2 Berlex Oncology Foundation Fellow. To whom requests for reprints should be addressed, at Division of Oncology (Mailstop: RM-17), 1959 Northeast Pacific Street, patients had advanced stage breast cancer in complete or partial remission with Health Science Building BB1321, Seattle, WA 98195. stable clinical condition at the time of collection. Patients were not actively 3 The abbreviations used are: DC, dendritic cells; APC, antigen-presenting cells; HPC, receiving chemotherapy. Briefly, patients were administered G-CSF at a dose hematopoietic progenitor cells; PBSC, peripheral blood stem cells; BM, bone marrow; of 10 pg/kg daily for 3 to 4 days to mobilize stem cells. Leukapheresis was G-ChF, granulocyte-colony-stimulatingfactor;GM-CSF,granulocyte-macrophage-colo ny-stimulating factor; FACS, fluorescence-activated cell sorting; TNF-a, tumor necrosis performed on day 3 or 4. Aliquots (3 ml) of the leukapheresis product were factor a; MLR, mixed leukocyte reaction; PE, phycoerythrin. used to purify CD34@HPC. 1099

Separation of CD34@ HPC and CD4@ or CD8@ T Lymphocytes. were used as responder cells. Dendritic cell cultures were harvested after CD34@ HPC were isolated from bone marrow or G-CSF-mobilized peripheral 12â€”16days, irradiated with 30 Gy, and added to the responder cells at different blood. CD4@ and CD8@ T lymphocytes were purified from peripheral blood concentrations (102-104/well). The assay was performed in 96-well round mononuclear cells or PBSC using the cell separation system Ceprate LC Kit bottomed plates (Corning, Coming, NY) at 37Â°C.The medium used consisted (CelIPro, Bothell, WA; Ref. 19). First, samples were processed using Ficoll of equal parts of EHAA (Biofluids) and RPMI 1640 (Gibco) with 10 mM Hypaque density gradient centrifugation (Pharmacia, Piscataway, NJ). Cells L-glutamine, 200 units/ml penicillin, 200 units/ml streptomycin, 2.5 X iO@ M obtained from the interface were washed and resuspended in PBS-1% BSA. In 2-mercaptoethanol, and 10% human serum (human AR CELLect; ICN Flow, order to purify CD34@ HPC, up to 108 cells/mI were incubated for 25 mm on Costa Mesa, CA). After 4 days, cells were pulsed with 50 pJ/well [3H]thymi ice with 40 @g'mlbiotinylated mAb anti-CD34 in PBS-1% BSA. Purification dine (1 mCi/rn]) for 8 h and counted. Data are shown as the mean of four of CD4@ and CD8@ I lymphocytes required a two-step labeling using 20 replicates. @g'mlanti-CD8 mAb or anti-CD4 mAb, respectively, for 25 mm on ice. Cells Antigen-presenting Assay. For induction of antigen-specific T cell re were washed with PBS-l% BSA and incubated with a biotinylated antimouse sponses two different antigens were used. Peptide p42â€”56,derived from the mAb for an additional 25 mm. The biotinylated cells were washed with amino acid sequence of HER-2/neu protein, is 15 amino acids in length and PBS-1% BSA to remove any unbound antibody. This fraction, in a volume of was chosen based on an increased probability of binding to Class II MHC 1 ml PBS-5% BSA/lO' cells, was applied to the Ceprate column, which molecules (17). The peptide was synthesized and purified by Dr. P. S. H. Chou contained sterile avidin-coated polyacrylamide beads. After washing with (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). and eluted with PBS. Dendritic cell cultures were harvested, spun, and resuspended in equal Purity of recovered CD4@ and CD8@ cells was 90% to >95% as determined amounts of EHAA and RPMI 1640 supplemented as described above. Differ by staining with anti-CD4 and anti-CD8 mAb, respectively. The selected I ent cell concentrations were loaded with tetanus toxoid protein (25 p@g/ml)or cells were not activated through the selection procedure. Purity of CD344 cells HER-2/neu p42â€”56 peptide (50 @g/ml). Following an incubation period for 1 depended on the source of the HPC. CD34@ cells isolated from bone marrow to 2 h at 37Â°C,the pulsed dendritic cell population was irradiated (30 Gy) and regularly showed a purity of 80% to >95%, whereas the purity from PBSC added to autologous CD4@ I lymphocytes that acted as responder cells derived CD34@ cells ranged from 30 to 80% depending on the response to (5 X 104/well).Proliferativeresponsewas measuredas a functionof thymidine G-CSF treatment of the individual patient. uptake and results are shown as the mean of four replicates. Dendritic Cell Culture Derived from HPC. DC were generatedusing a modified method, described by Caux et al. (14), for generating DC from RESULTS umbilical cord blood. All cell cultures were initiated from CD34@ HPC, which had been isolated through positive selection by immunoaffinity columns. The DC Can Be Generated from CD34@ HPC from BM and Pe purified cells were resuspended in RPM] 1640 medium (GIBCO, Grand Island, ripheral Blood in the Presence of GM-CSF and TNF-a. Initial NY) containing 2.5 X i0@ mM 2-mercaptoethanol, 100 units/mI penicillin, experiments asked whether the methods developed by Caux et a!. (14) 100 @Wmlstreptomycin, 2 mM L-glutamine, and 10% FCS. The culture for growing DC from cord blood could induce the growth of DC from medium was further supplemented with GM-CSF (100 ng/ml) (Schering adult bone marrow. CD34@ HPC from BM were enriched by positive 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) selection (90% purity) and cultured with GM-CSF plus TNF-a for 12 at a final concentration of i0@ cells/ml/well and split every 4â€”5days. After to 16 days. Results showed that the total cell number increased 12â€”16days of culture time, cells were harvested and used for phenotyping and approximately 40-fold, ranging from 20- to 80-fold (Fig. 1A). During functional assays. cell growth the CD34@ HPC population lost the CD34 marker which Monocyte Culture. Monocytes were enriched as described previously (2). was undetectable by day 9, and cells with a more differentiated In short, PBSC (2 x l06/ml) were cultured in supplemented RPMI 1640 in morphology appeared (Fig. 1B). In order to follow the differentiation Petri dishes (100 mm; Falcon, Lincoln Park, NJ). After 36 h of culture at 37Â°C, of CD34@ HPC into DC, the CD1a molecule, known as a marker for plates were washed three times in order to remove the nonadherent cells. Cells dendritic Langerhans cells, was used. CD1a expression gradually that remained adherent or that readhered to tissue culture plastic after 36 h increased, reaching a maximum level on day 15 (20). At that time were used as an enriched source of monocytes. In FACS analysis, 70% of the point the cultured population was heterogeneous, and the CD1a mol adherent cells displayed the monocyte marker CD14. Flow Cytometric Analysis. Ihe following mAbs were used for FACS ecule was displayed by 30â€”60% of the cells, depending on the analyses: anti-CD1a, anti-CD34 (Becton Dickinson, San Jose, CA); anti-CD8O individual cell culture. The relative number of CD1a expressing cells (B7/BB1) (provided by Dr. E. Clark, University of Washington, Seattle, WA); anti-CD35 (complement receptor CR1; ACCU, Westbury, NY); anti-CD64 200 (FcyRI; Medarex, Annandale, NJ), PE-conjugated anti-CD1a, anti-CD3, anti 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 80 1 50 ment goat anti-mouse IgG (Zymed, San Francisco, CA). â€”.-â€”COlaCD34 0 B Double-color fluorescence staining of cell cultures was carried out by K 0 sequential incubation of mAbs. Briefly, cells were resuspended in PBS-1% a 2 100 :n> BSA and seeded into microtiter plates at a final concentration of iO@cells/well. E 0) z 40 In order to reduce nonspecific binding, 10 @lgoatimmunoglobulin (3 mg/mI) a@ a were added to each well. After 10 mm, cells were incubated with the uncon 0 at6 50 jugated mAbs at saturating concentrations for 10 mm on ice, washed twice 20 with 150 @.dPBS-l% BSA and stained with FIIC-F(ab')2 fragment conjugated goat anti-mouse IgG for an additional 20 mm. Following two washes with 0â€”Â°@â€” PBS-1% BSA, 10 pi mouse immunoglobulinswere added for 10mm to reduce 0 9 11 15 19 cross-reactivity. Finally, cells were incubated with PE-labeled mAb for 20 mm, 1 5 9 11 15 19 1 5 washed twice with PBS-1% BSA and fixed with 1% paraformaldehyde. Days in Culture Days in Culture Negative controls were performed with FITC-F(ab')2 goat anti-mouse IgG and Fig. 1. Kinetics (A) and phenotype (B) of dendritic cell generation from BM-derivcd a PE-conjugated unrelated murine mAb (Becton Dickinson). Fluorescence 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 analyses were performed with a FACScan flow cytometer (Coulter). during cell growth. Expression of CD1a, a marker related to DC, increased during culture, MLR. Naive CD4@or CD8@I lymphocytes (5 X 104/well) isolated reaching a maximum level on day 15. Decreasing CD1a expression after day 15 is related through the Ceprate LC Kit from normal donor peripheral blood lymphocytes to the expanding monocytes. 1100 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. @@@ ; -

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 (80â€”90%)were positive for CD4, which is known to be expressed by cultured DC (20). Subpopulations of CD1a@ cells (30â€”50%) were positive for the monocyte marker CD14, which indicates that the @ @f> CD1a@ population can subdivide into CD1a@/CD14â€” 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

@ @iyJ. @Â° T cells. After 12â€”14days of culture, cells generated from BM or mobilized peripheral blood HPC-derived CD34@ HPC were used as

Fig. 2. Morphology of adherent cells generated from CD34@ HPC in response to CD1a CD4 B7/BB1 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@cellswerederivedfroma startingpopulationof @ 2 X 10@CD34@ HPC. HLA-DR CD14 CD64 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 L.J'@'\:@@@ era] blood-derived CD34@ HPC were, in general, less pure (30â€”80%) than BM-derived HPC (80 to >95%) after positive selection and expanded to a lesser degree (5â€”20-fold)proportional to the percentage @ of CD34@ cells in the initial culture. C035 c03 @D8 Cultured Cells Derived from CD34@ HPC Display Heterogene ous Morphology and Phenotypic Markers of DendriticlLanger @ hans Cells and Monocyte/Macrophages Including the Expression /@:\

@ of Class II MHC and Costimulator CD8O (B7IBB1) Molecules. I} â€˜\\ I @@ The morphology of cultured cells was evaluated at days 12â€”14. @L-@ @L-:-@ â€˜â€¢@-_@-- During 2 weeks of culture the homogeneous population of small mononuclear cells differentiated into a heterogeneous population of @ C019 C056 CD34 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, I\ typical features of monocytes and macrophages. The second major L/@@ cell population consisted of small, round, and nonadherent cells without vacuoles or projections which represented about 30% of the 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 cell culture. This morphologically defined second cell population isotype-matched nonreactive control mAb; bold lines, staining with mAb as indicated. The could not be categorized as a known functionally differentiated cell cell cultures were positive for CD1a, CD4, CD8O(B7/BBI), HLA-DR (Class II MHC), type. The third and smallest population (20%) contained mostly CDI4, CD64 (Fc'yRI),and CD35 (complement receptor CR1) and were negative for CD3, CD8, CD19, CD56, and adherent cells with a dendritic cell morphology with long delicate membrane projections (Fig. 2). The cells with dendritic morphology DCSurfaceTable 1Phenotype of HPC-derived were generally overgrown by typical macrophages after 2 weeks. Immunofluorescence analyses were performed between days 12 DCCD1al0-.60@'CD410-90CD1420-80CD8O10-60HLA-DR50-90CD34<5CD3<5CD8<5CD19<5CD56<5antigensHPC-derived and 15 with BM- and peripheral blood-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. a Cell surface phenotype of BM and peripheral blood-derived CD34@ cells varied Results were similar for populations generated from BM or peripheral between cultures. The range of cell surface marker expression for CD1a, CD4, CD14, bloodHPC. 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 To characterize the cell populations in more detail, double-color CD34 purity. flow cytometric analyses were performed using CD1a as a reference b Percentage of positive cells. 1101

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. GENERA11ONOFIMMUNOS11MULATORYDENDRI11CCELLS stimulator cells for allogeneic CD4@ as well as CD8@ T lymphocytes 8000 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 #@Â° 6000 and the number of cultured DC as stimulator cells varied. The level of E T cell proliferation was dependent on the number of DC/well. The T 0@ C-) cells were not observed to be activated by the method of purification used, as determined by a lack of thymidine incorporation without a, stimulator cells added. DC Generated from CD344 HPC Can Present Antigens to I 4000@ 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 2000@ 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 p42â€”56,was a 15-amino acid peptide derived from the normal sequence of HER-2/ 0@ neu, a protein which is overexpressed in many breast cancer patients. As the number of DC/well increased, the proliferative response of 0 1 10 100 1000 10000 CD4@ T lymphocytes to tetanus toxoid and HER-2/neu peptide was observed to increase. In this patient, the response to tetanus toxoid Number of stimulator cells per well was greater than the response to HER-2/neu peptide. Of note, DC alone at higher numbers induced a T cell response presumably re Fig. 6. Antigen presentation by dendritic cell cultures. Antigen-pulsed PBSC-dcrlved flecting an autologous MLR. In a second patient, the capacity of DC DC from a breast cancer patient induced a protein- or peptide-specific CD4@response. HER-2/neu p42â€”56peptide, @,tetanus toxoid protein, t no antigen, A. generated from CD34@ HPC to present peptides was compared to

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 a proliferate. Again, DC alone stimulated T cell proliferation. In three of the five patients tested, a proliferative response to HER-2/neu could not be detected. B7/BB1 CD1a COla 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 DISCUSSION subpopulation of CDIa cells was also positive for CD14. 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 CD4 resulted in an approximately 40-fold expansion of the starting cell number over 2 weeks and the generation of CD34-negative DC in a 10000 number equivalent to at least the number of starting CD34@ cells. E CD34@ HPC derived from either G-CSF-mobiized peripheral blood 0. U or BM are capable of acting as the starting population. The advantages a, .@ 1000 of using BM over PBSC are a greater purity of CD34@ stem cells (5 0. following cell separation (80 to >95% versus 30â€”80%), and subse

.@ quent better cell growth (20â€”80-fold versus 5â€”20-fold). The col lection of PBSC results in a higher total stem cell number after 100 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 0 1 10 100 1000100000 1 10 100 100010000 purity CD34 preparations from the peripheral blood indicates that DC Number of stimulator cells per well cultures are derived mainly from CD34@ cells. This hypothesis is Fig. 5. MLR using BM-derived dendritic cell cultures as stimulator cells and aliogeneic supported by single-cell experiments, which revealed that DC cob CD4@and CD8@T lymphocytes as responder cells. T lymphocyte proliferation can be nies originate from CD34@ cells of the BM, as recently reported by 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 Reid et aL (21, 22). alloreactivity. The rationale for using TNF-cs in addition to GM-CSF was drawn 1102

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. GENERATION OF IMMUNOSTIMULATORY DENDRITIC CELLS 5000 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 â€œden dritic cellsâ€•or whether some DC are CD1a@/CD14@. Others have shown that DC can be sorted from dimly stained CD14@ cells (34). I 4000 I An important issue not addressed is possible functional differences in 0@ antigen presentation between the defined phenotypic populations. C-, 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 4@) 0@ was consistently shown using DC cultures derived from either bone marrow (4/4) or mobilized peripheral blood HPC (6/6). In contrast,

@0 the ability to stimulate protein- and peptide-specific responses was F- less consistent. In the experiments presented, cultured DC effectively @ 2000 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. 1000@ 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 0 issue that remains is identifying culture conditions that expand DC Dendritic cells Monocytes which consistently retain the ability to process and present exogenous antigens. Other studies, as well as the data presented here, demon Fig. 7. Comparison of dendritic cell cultures and monocytes, from a breast cancer strate augmentation of MLR with human DC derived from progenitor patient, as APC. DC and monocytes were loaded with HER-2/neu p42â€”56(R)or no antigen (Cl). Peptide-pulsed PBSC-derived DC induced a specific CD4@ response to cells (14, 38). Results from our laboratory, however, show that cx HER-2/neu p42â€”56peptide,whereas monocytes from the same patient were not able to trapolation of dendritic cell function from MLR to specific antigen present the peptide to the autologous @T@+Tlymphocytes. presenting capability is not necessarily appropriate. Although function in a MLR is reproducible, these same DC would have variable from the experience of others who showed that TNF-a modifies function when presenting specific antigens. Subsequent studies should GM-CSF-induced myelopoiesis and facilitates the development of DC focus on the interrelationship between cell growth and the functional from cord blood CD34@ HPC (14). Using this regimen to culture ability to stimulate protein- and peptide-specific responses and should positively selected CD34@ HPC from BM or peripheral blood resulted utilize antigen-presenting function rather than cell number or alloge in a heterogeneous population of three major cell types based on neic MLR for optimization of culture conditions. The availability of morphology: DC, monocytes, and small round cells of unknown sufficient numbers of efficient antigen-presenting DC should facilitate function. The DC were mostly adherent and displayed a typical the study of T cell-mediated responses to tumor-associated antigens. morphology with delicate membrane projections. The monocytes were largely adherent and vacuolated and formed a tight cell network ACKNOWLEDGMENTS which tended to overgrow the DC after 2 weeks. Both DC and monocytes were expanded under the same conditions. It is possible We thank Ed Clark for providing anti-CD8OmAb B7/BB1, Kirsten Stray, that they may share the same precursor, a concept recently presented Sandra R. Emery, and Faith Shiota for technical assistance, and Kevin by Reid et a!. (21, 22), and is supported by the observation that fully Whitham for manuscript preparation. In addition, we thank the members of the differentiated monocytes could be a source for generating veiled autologous bone marrow transplant team, leukapheresis unit, and cryopreser vation unit at the Fred Hutchinson Cancer Research Center for supplying the accessory cells when cultured in serum-free medium (23, 24). Future peripheral blood stem cells used in these studies. attempts at generating DC from CD34@ HPC should explore the influence of other cytokines to drive HPC differentiation into a more REFERENCES homogeneous population of DC. Cytokines of interest include inter leukin 1 which signals interleukin 1 receptor to up-regulate GM-CSF 1. Steinman, R. M., and Cohn, Z. A. Identification of a novel cell type in peripheral receptors on epidermal DC; interleukin 4 which drives monocytes into lymphoid organs of mice. J. Exp. Med., 137: 1142â€”1162,1973. 2. Young, J. W., and Steinman, R. M. Dendritic cells stimulate primary human cytolytic cells with dendritic processes, down-regulates monocyte markers, and lymphocyte responses in the absence of CD4@ helper T cells. J. Exp. Med., 171: enhances antigen-presenting capacity; and interleukin 6 which stim 1315â€”1332,1990. 3. Steinman, R. M. The dendritic cell system and its role in immunogenicity. Annu. Rev. ulates monocyte-derived Langerhans cells via autocrine cytokine Immunol., 9: 271â€”296,1991. production (24â€”28). 4. Sornasse, T., Flamand, V., Becker, 0. D., Bazin, H., Tielemans, F., Thielemans, K., The cell populations derived by culture of CD34@ HPC lost the Urbain, J., Leo, 0., and Moser, M. Antigen-pulsed dendritic cells can efficiently induce an antibody response in vivo. J. Exp. Med., /75: 15â€”21,1992. CD34@ marker, were positive for markers known to be expressed by 5. Paglia, P., Girolomoni, G., Robbiati, F., Franucci, F., and Ricciardi-Castagnoli, P. DC and monocytes (CD1a, CD4, CD8O, and Class II MHC; Refs. 3, Immortalized dendritic cell line fully competent in antigen presentation initiates 14, 29â€”33),and lacked surface markers for B, T, and natural killer primary T cell responses in vivo. J. Exp. Med., 178: 1893â€”1901,1993. 6. Levin, D., Constant, S., Pasqualini, T., Flavell, R., and Bottomly, K. Role of dendritic cells (CD3, CD19, CD8, and CD56). The studies focused particularly cells in the priming of CD4@T lymphocytes to peptide antigen in vivo.J. Immunol., on the CD1a molecule which is associated with the immunostimula 151: 6742â€”6750,1993. 7. Bhardwaj, N., Young, J. W., Nisanian, A. J., Baggers, J., and Steinman, R. M. Small tory cell fraction from HPC-derived cell cultures (14). Double-color amounts of superantigen, when presented on dendritic cells, are sufficient to initiate immunofluorescence showed that the majority of CD1a@ cells ex T cell responses.J. Exp. Med., 178: 633â€”642,1993. 1103

8. Katz,S. I.,Tamaki,K., andSachs,D. H. EpidermalLangerhanscellsarederivedfrom 23. Peters, I. H., RuhI, S., and Friedrichs, D. Veiled accessory cells deduced from cells originating in bone marrow. Nature (Lond.), 282: 324â€”326,1979. monocytes. Immunobiology, 176: 154â€”166,1987. 9. Huang, A. Y. C., Golumbek, P., Ahmadzadeh, M., Jaffee, E., Pardoll, D., and 24. Rossi, 0., Heveker, N., Thiele, B., Gelderblom, H., and Steinbach, F. Development Levitsky, H. Role of bone marrow-derived cells in presenting MHC class I-restricted of Langerhans cell phenotype from peripheral blood monocytes. Immunol. Left., 31: tumor antigens. Science (Washington DC), 264: 961â€”965,1994. 189â€”198,1992. 10. Knight, S. C., and Stagg, A. J. Antigen-presenting cell types. Curr. Opin. Immunol., 25. Peters, J. H., Ruppert, J., Gieseler, R. K. H., Najar, H. M., and Xu, H. Differentiation 5: 374â€”382,1993. of humanmonocytesintoCD14negativeaccessorycells:do dendriticcellsderive 11. Voorhis, W. C. V., Valinsky, J., Hoffman, E., Luban, J., Hair, L S., and Steinman, from the monocytic lineage? Pathobiology, 59: 122â€”126,1991. R.M.Relativeefficacyofhumanmonocytesanddendriticcellsasaccessorycellsfor 26. Ruppert, J., Friedrichs, D., Xu, H., and Peters, J. H. IL-4 decreases the expression of T cell proliferation.J.Exp. Med., 158: 174â€”191,1983. the monocytedifferentiationmarkerCD14,paralleledby an increasingaccessory 12. Thomas, R., Davis, L. S., and Lipsky, P. E. Comparative accessory cell function of potency. Immunobiology, 182: 449â€”464,1991. human peripheral blood dendritic cells and monocytes. J. Immunol., 151: 6840â€” 27. Sallusto, F., and Lanzavecchia, A. Efficient presentation of soluble antigen by 6852, 1993. cultured human dendritic cells is maintained by granulocyte/macrophage colony 13. DeBruijn, M., Schumacher, T., Nieland, J., Ploegh, H., Kast, W., and Meief, C. stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor a. J. Peptide loading of empty major histocompatibility complex molecules on RMA-S Exp. Med., 179: 1109â€”1118,1994. cells allows the induction of primary cytotoxic T lymphocyte responses. Eur. J. 28. Kampgen, E., Koch, F., Heufler, C., Eggert, A, Gill, L L, Gills, S., Dower, S. K., Immunol., 21: 2963â€”2970,1991. Ramoni, N., and Schuler, 0. Understanding the dendritic cell lineage through a study 14. Caux, C., Dezutter-Dambuyant, C., Schmitt, D., and Banchereau, J. GM-CSF and of cytokine receptors. J. Exp. Med., 179: 1767â€”1776,1994. TNF-alpha cooperate in the generation ofdendritic Langerhans cells. Nature (Lond.), 29. Young, J. W., Koulova, L, Soergel, S. A, Clark, E. A., Steinman, R. M., and Dupont, 360: 258â€”261,1992. B. The B7/BB1 antigenprovidesone of severalcostimulatorysignalsfor the activa 15. Paik, S., Hazan, R., Fisher, E., 5ass, R., Fisher, B., Redmond, C., Schlessinger, J., tion of @T@+Tlymphocytes by human blood dendritic cells in vitro. J. Clin. invest., Lippman, M., and King, C. Pathologic fmdings from the National Surgical Adjuvant 90: 229â€”237,1992. Breast and Bowel Project: prognostic significance of erbB-2 protein overexpression 30. Larsen, C. P., Ritchie, S. C., Pearson, T. C., Linley, P. 5., and Lowry, R. P. Functional in primary breast cancer. J. Clin. Oncol., 8: 103â€”112,1990. expression of the costimulatory molecule, B7/BB1, on murine dendritic cell popula 16. Ioannides, C., loannides, M., and O'Bnan, C. T cell recognition of oncogene prod tions. J. Exp. Med., 176: 1215â€”1220,1992. ucts: a new strategy for immunotherapy. Mol. Carcinog., 6: 77â€”82,1992. 31. Kosco-Vilbois, M. H., Gray, D., Scheidegger, D., and Julius, M. Follicular dendritic 17. Disis, M. L, Calenoff, E., McLaughlin, 0., Murphy, A. E., Chen, W., Groner, B., cells help resting B cells to become effective antigen-presenting cells: induction of Jeschke, M., Lydon, N., Mcfllynn, E., Livingston, R. B., Moe, R., and Cheever, M. A. B7IBB1 and upregulation of major histocompatibility complex class II molecules. J. Existent T cell and antibody immunity to HER-2/neu protein in patients with breast Exp. Med., 178: 2055â€”2066,1993. cancer. Cancer Res., 54: 16â€”20,1994. 32. O'Doherty, U., Steinman, R. M., Peng, M., Cameron, P. U., Gezelter, S., Kopeloff, 18. Disis, M. L, Smith, J. W., Murphy, A. E., Chen, W., and Cheever, M. A. In vitro I., Swiggard,W. J., Pope, M., andBhardwaj,N. Dendriticcells freshlyisolatedfrom generation of human cytotoxic T-cells specific for peptides derived from the HER human blood express CD4 and mature into typical immunostimulatory dendritic cells 2/neu protooncogene protein. Cancer Res., 54: 1071â€”1076,1994. after culture in monocyte-conditioned medium. J. Exp. Med., 178: 1067-1078, 1993. 19. Shpall, E. J., Jones, R. B., Bearman, S. I., Franklin, w. A., Archer, P. 0., Curiel, T., 33. Porcelli, S., Morita, C. T., and Brenner, M. B. CD1b restricts the response of human Bifter, M., CIm@an, H. N., Stemmer, S. M., Purdy, M., Myers, S. E., Hami, L, Taffs, CD4â€”8â€”Tlymphocytes to a microbial antigen. Nature (Lond.), 360: 593-597, 1992. S., Heimfeld,S., Hallagan,J., and Berenson,R. J. Transplantationofenriched 34. Thomas, R., Davis, L S., and Lipsky, P. E. Rheumatoid synovium is enriched in CD34-positive autologous marrow into breast cancer patients following high-dose mature antigen-presenting dendritic cells. J. Immunol., 152: 2613â€”2623,1994. chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth 35. Young, J. W., and Steinman, R. M. Accessory cell requirements for the mixed factos on engrafment. J. Chin.Oncol., 12: 28â€”36,1994. leukocyte reaction and polyclonal mitogens, as studied with a new technique for 20. Wood, 0. S., Warner, N. L, and Warnke, R. A. Anti-Lcu-3/T4 antibodies react with enriching blood dendritic cells. Cell. ImmunoL, 111: 167â€”182,1988. cells of monocyte/macrophage and Langerhans lineage. J. Immunol., 131: 212â€”216, 36. Streilein, J. W., and Grammer, S. F. In vitro evidence that Langerhans cells can adopt 1983. two functionally distinct forms capable of antigen presentation to T lymphocytes. J. 21. Reid, C. D. L, Fryer, P. R., Clifford, C., Kirk, A., Tikerpae, J., and Knight, S. C. Immunol., 143: 3925â€”3933,1989. Identification of hematopoietic progenitors of macrophages and dendritic Langerhans 37. Romani, N., Koide, S., Crowley, M., Wittner-Pack, M., Livingstone, A. M., Fathman, cells (DL-CFU) in human bone marrow and peripheral blood. Blood, 76: 1139â€”1149, C. 0., Inaba,K., andSteinman,R. M. Presentationofexogenousproteinantigensby 1990. dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal 22. Reid, C. D. L., Stackpoole, A., Meager, A, and Tikerpae, J. Interactions of tumor Langerhans cells. J. Hip. Med., 169: 1169â€”1178, 1989. necrosis factor with granulocyte-macrophage colony stimulating factor and other 38. Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A, Trockenbacher, B., cytokines in the regulation of dendritic cell growth in vitro from early bipotent Konwalinka, G., Fritsch, P. 0., Steinman, R. M., and Schuler, G. Proliferating Q@34+progenitors in human bone marrow. J. Immunol., 149: 2681â€”2688,1992. dendritic cell progenitors in human blood. J. Exp. Med., 180: 83â€”93,1994.

1104

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. Generation of Immunostimulatory Dendritic Cells from Human CD34+ Hematopoietic Progenitor Cells of the Bone Marrow and Peripheral Blood

Helga Bernhard, Mary L. Disis, Shelly Heimfeld, et al.

Cancer Res 1995;55:1099-1104.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/5/1099

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/55/5/1099. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.