[cancer research 59, 2150–2158, may 1, 1999] …[cancer research 59, 2150–2158, may 1, 1999]...

[CANCER RESEARCH 59, 2150–2158, May 1, 1999]

Presentation of Tumor Antigens by Phagocytic Dendritic Cell Clusters Generatedfrom Human CD341 Hematopoietic Progenitor Cells: Induction of AutologousCytotoxic T Lymphocytes against Leukemic Cells in Acute MyelogenousLeukemia Patients1

Shin-ichiro Fujii, Koji Fujimoto, Kanako Shimizu, Taichi Ezaki, Fumio Kawano, Kiyoshi Takatsuki,Makoto Kawakita, and Kenjiro Matsuno 2

The Center for Bone Marrow Transplantation and Immunotherapy, Institute for Clinical Research, Kumamoto National Hospital, Kumamoto 860-0008 [S-i. F., K. F., F. K.];Second Department of Anatomy [K. S., T. E., K. M.] and Second Department of Internal Medicine [M. K.], Kumamoto University School of Medicine, Kumamoto 860-0811;Kitano Hospital Tazuke Kofukai Medical Research Institute, Osaka 530-0026 [K. T.], Japan

ABSTRACT

The use of antigen-presenting dendritic cells (DCs) is currently pro-posed for tumor immunotherapy through generation of CTLs to tumorantigens in cancer patients. In this study, DCs were differentiated usinggranulocyte-macrophage colony-stimulating factor and tumor necrosisfactor-a from CD341 hematopoietic progenitor cells that had been mo-bilized into the peripheral blood. To use the phagocytic activity of DCs forprocessing and presentation of tumor antigens, we established DC clusterscontaining immature DCs by preserving proliferating cell clusters withoutmechanical disruption. After an 11-day culture, the developed clusterscontained not only typical mature DCs but also immature DCs thatshowed active phagocytosis of latex particles, suggesting that the clustersconsisted of DCs of different maturational stages. These heterogeneousclusters could present an exogenous protein antigen, keyhold limpet he-mocyanin, to both CD41 and CD81 T lymphocytes. Furthermore, in threeacute myelogeneous leukemia patients, clusters pulsed with autologousirradiated leukemic cells could also induce antileukemic CTLs. The me-chanical disruption of clusters abrogated the induction of CTLs to leuke-mic cells as well as to hemocyanin. This observation gives an importantinformation for the use of heterogeneous DC clusters derived from autol-ogous peripheral blood CD341 cells in the case of immunotherapy forleukemia.

INTRODUCTION

Allogeneic bone marrow transplantation in leukemia is well estab-lished. Its efficacy may result from not only high-dose chemotherapybut also an allogeneic immune mechanism, known as the GVL3 effect(1, 2). Currently, autologous PBSCs are used as an alternative sourceof stem cells for hematopoietic regeneration. When autologousPBSCT was performed in leukemic patients, however, it had a highrelapse rate, which is principally connected with the lack of a GVLeffect and with the presence of clonogenic malignant cells in themarrow graft (1). Attempts to develop the autologous immunogeniceffect on PBSCT have been made, and some of them have been foundto be effective. For example, modified immunotherapies together withPBSCT, such as administration of recombinant interleukin 2 or lym-

phokine-activated killer cells (3, 4), and cyclosporine-induced autol-ogous graft-versus-host disease have been reported to successfullytreat patients with lymphoma and other malignancies (5). This impliesthat a proper immunological treatment of leukemic patients before orafter PBSCT may induce a GVL effect, making PBSCT therapy inmalignancy more successful and profitable.

Induction of tumor-specific CTLs usually requiresin vivo primingwith tumor cells. In fact, CTL effectors can be isolated from patientswith AML or chronic myelocytic leukemia (6), but the generation ofsuch cellsin vitro from naive precursors has not been reported. DCsare the most potent professional APCs. DCs in the periphery captureand process antigens, migrate to lymphoid organs, and initiate im-mune responses (7). Therefore, DCs preloaded with leukemia antigensmight be able to induce CTLs from autologous T cells. For processingparticulate antigens, such as leukemic cells, phagocytic activity ofDCs may be essential. Although DCs were regarded as nonphago-cytic, DCs have been recently demonstrated to phagocytose andprocess antigens at the immature stage in mice and rats (8–12).

Here, we established a method to generate DC clusters containingimmature cells with phagocytic activity from human HPCs and in-vestigated whether or not they could induce specific CTLs againstleukemic cells. (a) We developed DC clusters from CD341 cellfraction of PBSCs by GM-CSF and TNF-a (13) and tested theirphagocytic activity. (b) We examined whether the developed DCspossessed antigen-presenting capability using KLH as an exogenousprotein antigen (14). (c) We investigated whether specific anti-leuke-mic CTLs could be induced by the DCs pulsed with irradiated autol-ogous leukemic cells during their development. Our study demon-strates that immunization within vitro generated DC clusters, whichare preloaded with leukemia antigens, can induce CTLs. This strategyprovides an attractive approach to developing novel vectors for cancertherapies.

MATERIALS AND METHODS

Antigens and Reagents.KLH and ovalbumin were purchased from SigmaChemical Co. (St. Louis, MO), and TNF-a was from Genzyme (Cambridge,MA). Recombinant human GM-CSF was kindly provided by Schering-Plough(Kenilworth, NJ). All cultures were performed in RPMI 1640 (Life Technol-ogies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated pooledhuman AB serum, 2 mM L-glutamine, 100 mg/ml kanamycin, and 100 units/mlpenicillin (hereafter designated as complete medium).

Source of Cells.CD341 HPCs were isolated from PBSCs of recombinanthuman G-CSF-treated leukemia patients who were scheduled to undergoautologous stem cell transplantation after obtaining informed consent. Allpatients were in complete remission with stable clinical condition at the timeof cell collection. After chemotherapy with Ara-C (100 mg/m2 for 7 days),busulfan (4 mg/kg for 4 days), VP-16 (20 mg/kg for 2 days), and Ara-C (6 g/m2

for 2 days), recombinant human G-CSF was administered at a dose of 10mg/kgdaily for 3–4 days. PBSCs were then harvested by leukapheresis using a Cobe

Received 11/16/98; accepted 3/4/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Public Trust Haraguchi Memorial Cancer ResearchFund and also partially by a grant for scientific research from Tsumura Co. Ltd. (Tokyo,Japan) and Matsushita Hospital (Kumamoto, Japan).

2 To whom requests for reprints should be addressed, at Second Department ofAnatomy, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan.Phone: 81-96-373-5045; Fax: 81-96-373-5048; E-mail: [email protected].

3 The abbreviations used are: GVL, graft-versus-leukemia; PBSC, peripheral bloodstem cell; PBSCT, PBSC transplantation; DC, dendritic cell; APC, antigen-presentingcell; HPC, hematopoietic progenitor cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF-a, tumor necrosis factor-a; KLH, keyhole limpet hemocyanin;G-CSF, granulocyte colony-stimulating factor; AML, acute myelogenous leukemia; BM,bone marrow; moAb, monoclonal antibody; MLR, mixed leukocyte reaction.

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cell separator (Cobe BCT Inc., Lakewood, CO) on day 10 or 11 after chem-otherapy. In one series of experiments, a healthy donor was treated withG-CSF, and CD341 HPCs were isolated in a similar fashion.

Primary leukemic cells from each patient were stored in liquid nitrogen atde novoleukemia stage. In patient A [AML (M1)], leukemic cells comprised80.8% of cells in BM with a translocation of t(9;11)(q22;23) and phenotype ofCD381, CD331, c-kit1, HLA class I1, HLA class II1. In patient B [AML(M2)], leukemic cells comprised 61.0% of cells in BM with a normal chro-mosome of [46XY] and phenotypes of CD381, CD331, c-kit2, CD71, HLAclass I1, HLA class II2. In patient C [AML (M2)], leukemic cells comprised36.4% of cells in BM with a translocation of t(8;21) and phenotypes of CD381,CD331, c-kit2, CD71, HLA class I1, HLA class II1.

Separation of CD341 HPCs. An aliquot (3 ml) of harvested PBSCs,diluted 1:2 in RPMI 1640, was applied to Ficoll-Hypaque (Pharmacia,Uppsala, Sweden) density gradient centrifugation. Cells obtained from inter-face were washed with serum-free medium and then allowed to adhere toculture dishes in complete medium for 2 h to remove monocytes. Nonadherentcells were then washed and resuspended in PBS supplemented with 1% BSA,100 mg/ml kanamycin, and 100 units/ml penicillin (hereafter designated asisolation buffer). CD341 cells were positively purified with anti-CD34 moAb561-coated M-450 Dynabeads (Dynal, Oslo, Norway) by incubating 43 107

beads with 2.53 107–5 3 107 cells/ml for 60 min at 4°C. After removingrosetted cells with a magnet, and the beads were detached from the cells withDETACHaBEADs CD34 (Dynal) according to the manufacturer’s instruc-tions. This yielded 856 10% pure viable CD341 cells.

Generation of DCs from CD341 Cell Culture. DCs were generated fromCD341 cell populations of PBSCs (Fig. 1) in complete medium supplementedwith GM-CSF (100 ng/ml) and TNF-a (2.5 ng/ml). CD341 cells were culturedin six-well plates (Coster, Cambridge, MA) at a final concentration of 105

cells/well in 2 ml of complete medium at 37°C in a humidified atmosphere of

5% CO2 (14). At 11 days of culture, harvested cells were examined formorphology, surface phenotype was determined by flow cytometry, and allo-stimulating activity was examined by MLR assays. In this study, developingDC clusters were kept undisturbed, and 2 ml of fresh medium containingcytokines were added to the culture only at day 5. For an inhibition experiment,cluster formation was interrupted by frequent pipetting daily from day 5 to day11 of culture.

Isolation of CD41 and CD81 T Lymphocytes and Monocytes fromPBSCs. The CD342 population of PBSCs, remaining after positive selectionof CD341 cells, was cryopreserved in liquid nitrogen. For T-cell isolation,CD342 PBSCs were thawed and incubated with immunomagnetic beadscoated with anti-CD4 or anti-CD8 moAbs (Dynabeads M-450, CD4, or CD8;Dynal) for 30 min on ice. After washing with isolation buffer, the positive cellswere collected and beads were detached by incubation with CD4 or CD8DETACHaBEADs (Dynal) at 25°C for 60 min. The purity for both CD41 andCD81 cells was 856 5%.

For isolation of monocytes, PBSCs (23 106/ml) were cultured in completemedium at 37°C in Petri dishes (100 mm; Falcon, Lincoln Park, NJ). After 2 h,cells that remained adherent were dislodged by 1% trypsin-EDTA for 5 minand collected as a monocyte-enriched population, 70% of which displayedmonocyte marker CD14. They were used for both antigen-presenting andtarget macrophages in CTL assay to KLH antigen.

Electron Microscopy. DCs were generated on a plastic sheet (Wako Pure-Chemical Ind., Tokyo, Japan) in the same culture condition as described aboveand harvested on day 11. After washing with PBS, cells on the plastic sheetwere directly fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2)for 18 h and postfixed in 2% osmium tetroxide for 1 h at 4°C. The cells weredehydrated through a graded series of ethanols, infiltrated in propylenoxide/Epon mixtures, and then embedded in Epon 812. Ultrathin sections were cutand electron-stained with uranyl acetate and lead citrate and examined by atransmission electron microscope, JEM 100CX (Nihon Densi, Tokyo, Japan).

Flow Cytometric Analysis. Surface marker analysis ofin vitro culturedcells was performed using a FACScan and Lysis II software (Becton Dickin-son, Mountain View, CA). We used the following FITC-conjugated moAbs: tohuman CD1a, CD11c, CD34, and CD58 (Immunotech, Marseilles, France);CD4, CD14, HLA-DR, and CD80 (Becton Dickinson); MHC class I (DAKO,Glostrup, Denmark); MHC class II (Ancell Corporation, Bayport, MN); andCD40 and CD86 (PharMingen, Hamburg, Germany). moAbs to CD83 (Ref.15; Dr. T. F. Tedder, Duke University, Durham, NC) and fascin p55 (Ref. 16;Dr. E. Langhoff, Massachusetts General Hospital, Boston, MA) were kindlydonated. Two-color analysis was performed by using phycoerythrin-conju-gated moAbs to CD1a and CD83 (Immunotech) and fluorescein-conjugatedmoAbs to HLA-DR, CD14, CD40, CD86, and CD1a.

In Vitro Phagocytosis of Latex Particles.FluoSpheres [carboxylate-mod-ified microspheres which emit green fluorescence, 0.03% (v/v), 1-mm diam-eter; Molecular Probes, Eugene, OR] were applied to 33 104 DCs at day 5culture in 200ml of RPMI and were cultured in a chamber slide (eightchambers/slide, Lab-Tek; Nunc Inc., Naperville, IL) at 37°C in 5% CO2 for 6days (Fig. 1). At day 11, the chambers were gently washed and adherent cellswere air-dried and processed for immunostaining. Some cultures were sub-jected to two-color fluorescence-activated cell sorting analysis for phyco-erythrin-conjugated anti-CD1a and FluoSpheres.

Immunocytochemistry. Single immunostaining for cytosmears of thechamber slides was performed by the indirect immunoalkaline phosphatasemethod and colored red with alkaline phosphatase substrate kit I (Vector red;Vector Laboratories, Inc., Burlingame, CA), as described previously (17).Because the reaction product of Vector Red substrate emitted red fluorescence,both moAb (red) and latex (green) fluorescence could be examined simulta-neously under epifluorescence microscope at a wavelength exciting FITC (490nm). In addition, negative cells were visualized by a transillumination with agreen filter.

Allogeneic Mixed Leukocyte Reaction (MLR). PBSCs (53 104 cells/100ml) were used as responder cells. As allogeneic stimulator cells, up to 23 105

cells/100ml of CD341 HPC-derived DCs (Fig. 1), peripheral blood mononu-clear cells, or PBSCs from healthy volunteers were irradiated with 30 Gy. Bothwere cocultured for 6 days in complete medium in 96-well U-bottomed culturemicroplates (Costar, Cambridge, MA) and [3H]thymidine (Amersham, Amer-sham, United Kingdom) incorporation was measured after 8-h pulse with 1mCi per well. Results are shown as mean cpm of triplicates.Fig. 1. A schematic flowchart of the method used in this study.

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AUTOLOGOUS INDUCTION OF CTLs TO AML CELLS BY DCs



Fig. 2. A andB, phase contrast micrographs of cultured CD341 cells with GM-CSF and TNF-a at day 3 (A) and day 11 (B). In the early stage, cells are fusiform and dispersed(A), but gradually they gather to form cell clusters (B).Scale bars, 100mm. C–F, fluorescent microfluorographs of cytosmears of CD341 cell cultures in which fluorescent latex wasapplied to this culture at day 5 and examined at day 11. After immunostaining for CD1a (C), CD54 (D), MHC class II (E), and p55 (F), areas of low cellularity were photographed.Note some CD1a1, CD541, MHC class II1, and p551 cells (red fluorescence,small arrows) ingested latex (green). Also note the stromal cell-like cells (large arrows), which ingestedlatex but were negative for CD1a (C) and CD54 (D).Scale bars, 20mm. G andH, phase contrast micrographs of T cells cocultured with the DC cluster after 1 (G) and 7 (H) dayswhen the cluster became very large. T cells were preferentially associated with the DC cluster and expanded in a vicinity of the cluster (arrows).Scale bars, 100mm.

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Antigen Pulsing of DCs. For induction of CTLs, antigens were added tothe DC culture at days 3 and 7, when DCs were still at an immature stage, andthen CD41 and/or CD81 T lymphocytes were added at day 11 when aconsiderable proportion of DCs had already matured (Fig. 1). As an exogenousprotein antigen, KLH, a large protein (Mr 3 3 106–7.53 106) to which mostindividuals have not been sensitized, was added to DC cultures (;13 106

cells/2 ml) derived from PBSCs of two healthy volunteers. As a control,autologous macrophages (PBSC-derived monocytes) into which KLH (5 mg/ml) was directly introduced by osmotic lysis of pinosomes were used as otherAPCs (14). As autologous leukemic antigens, AML cells from three patientswere irradiated with 70 Gy and added to the corresponding autologous DCcultures at a cell ratio of 3:1.

Generation of CTLs. Autologous CD41 or CD81 T cells in PBSCfraction, which showed naive phenotype of CD45RA1 (.85%) andCD45RO2 (.95%), were used as responder cells. At day 11, the antigen-pulsed DC cultures were gently pipetted without disruption of the cluster,and medium containing free cells was removed. To this, a total of 43 106

purified CD41 or CD81 T cells in 2 ml of complete medium containing 10units/ml interleukin 2 was added (Fig. 1). After further culture for 7days, thesein vitro primed T cells were harvested and examined for CTLactivity.

CTL Assay. 51Cr-release assays were performed as described (18).Briefly, 10,000 target cells were labeled with Na2

51CrO4 (100mCi/2 3 106

cells) and were cultured with effector cells at various E:T ratios in triplicatein U-bottomed microtiter wells for 4 h at 37°C. As targets for KLH-specificCTLs, macrophages (PBSC-derived monocytes) were activated by precul-ture with macrophage colony-stimulating factor for 16 h and then pulsedwith KLH (5 mg/ml) or ovalbumin (5 mg/ml) for another 16 h. Unpulsedmacrophages with or without addition of free KLH (5 mg/ml) during CTLassay were also used. For antileukemic CTLs, autologous AML cells weretreated as targets in a similar manner as above. The spontaneous releasenever exceeded 20%.

Blocking Experiment. To investigate CD8 and MHC class I or CD4 andMHC class II restriction in the recognition of the target cells, blockingstudies were performed (14). Anti-CD4 or anti-CD8 moAbs (Becton Dick-inson) were added at the final concentration of 20mg/ml to effector cellsfor 30 min before coculture with target cells and to the coculture for the51Cr release assay. Either anti-MHC class I (Becton Dickinson) or anti-class II (Ancell Corporation, Bayport, MN) moAbs were added to targetcells in a same manner as above.

RESULTS

Characterization of DCs Generated from CD341 HPCs ofPBSC Fraction in the Presence of GM-CSF and TNF-a. WhenCD341 HPCs were cultured with GM-CSF plus TNF-a for 11days, the total number of cells increased;20-fold, ranging from10- to 50-fold (10 independent cell cultures). The extent of cellgrowth strongly correlated with the degree of purity of the startingCD341 cell population. Culture of CD341 HPCs produced largecells with polygonal, fusiform, or dendritic shape, which formedcell clusters by day 11 (Figs. 2,A and B, and 3). As shown by atransmission electron microscopy (Fig. 3), cells in the clustergenerally have an irregular euchromatic nucleus and a cytoplasmwith extremely long ruffled projections. However, no distinctjunctional specializations have as yet been noted at the contactpoints through these projections in these cell clusters. Mitotic cellswere often observed within the clusters, and the number and size ofthe clusters increased with time.

Because cell clusters could be dispersed by pipetting with relativeease, isolated cells were analyzed by flow cytometry (Fig. 4). Devel-oped cells contained different proportions of cells positive for DCmarkers, namely, CD1a, CD4, CD54, CD86, and HLA-DR (MHCClass II). Although some macrophages (CD141) were present as well,very few cells displayed cell surface markers of HPCs (CD34), B cells(CD19), T cells (CD3, CD8), or natural killer cells (CD56). In thisexperiment, 1.53 106 6 0.2 3 106 CD1a1 DCs were derived froma starting cell number of 23 105 CD341 HPCs. By double-color flowcytometric analyses using CD1a as a reference molecule (Fig. 5), itwas found that 16–40% of CD1a1 cells coexpressed HLA-DR,CD40, and CD83, indicating that these clustering cells containedtypical mature DCs.

When the phagocytic activity of the DC clusters was examined,;25%of the cells showed active ingestion of latex particles. By immuno-staining, phagocytic cells were found to be mostly CD1a2 or CD1a1 butCD11c1, CD541, MHC class II1/2 or MHC class II1, and fascin1 (Fig.2, C–F). FACS analysis showed that 25.96 2.5% of cells in the DCclusters were actively phagocytic and 19.96 3.2% of CD1a1 cells

Fig. 3. Transmission electron micrograph of acluster of DCs generated from a CD341 cell cultureat day 11. Their nuclei usually have euchromatin-rich nucleoplasm and a prominent nucleolus. Thecytoplasm contains abundant small membrane-bounded phagosomes, multivesicular bodies, smallvesicles, rough endoplasmic reticulum, and mito-chondria. Note that cells in the cluster generally havean eccentric irregular nucleus and a cytoplasm withextremely long ruffled projections. In the cluster,however, cells are only loosely packed in close con-tact with adjacent cells by these projections and nodistinct junctional specializations have been found atthe contact points. Also note that stromal cell-likeflat cells are underlying at the bottom of cell clusters(arrowheads). These cells have also long ruffledprojections but have a relatively darker cytoplasmwith fewer organelles except for mitochondria.Scalebar, 2 mm.

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ingested latex particles. These results indicated that phagocytic cellscorresponded to the relatively immature DC population.

The clusters seemed to grow upon a sheet of the flat cells spreading onthe plastic substratum, like stromal cells in bone marrow cell cultures(Fig. 3). They ingested a small amount of latex particles that accumulatedin the cytocenter (Fig. 2,C and D). By immunostaining, they werenegative or very weakly positive for CD1a (Fig. 2C), CD4, CD11c,CD14, CD54 (Fig. 2D), or CD80. We assume that these stromal cell-likecells do not belong to either lymphoid, macrophage, or DC lineages.These results revealed that cells forming the clusters were heterogeneousand mainly consisted of DCs in different maturational stages.

Capacity of the Cultured DCs to Stimulate the MLR. As shownin Fig. 6, CD341 PBSC-derived cells were potent stimulators againstresting T lymphocytes in primary allogeneic MLR, being more potentthan the allogeneic mononuclear cells or allogeneic PBSCs. These cellstherefore have functional as well as phenotypic characteristics of DC.

Induction of CTLs by DCs Pulsed with a Complex ProteinAntigen in Vitro. DCs but not macrophages pulsed with KLH couldinduce CD41 or CD81 T cells that efficiently lysed target KLH-pulsed macrophages (Fig. 7a). Nonpulsed DCs did not induce specificCTL activity (,5% at an E:T ratio of 50:1). Supernatants fromcocultures of KLH-pulsed DC and T cells were not specifically

Fig. 4. Surface phenotype of cells in clusters generatedfrom CD341 cell cultures at day 11. They contained differ-ent proportions of cells positive for DC markers, namely,CD1a, CD4, CD54, CD86, and HLA-DR. Although somemacrophages (CD141) were present as well, very few cellshad markers for HPCs (CD34), B cells (CD19), T cells(CD3, CD8), or natural killer cells (CD56).

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cytotoxic (,5% at an E:T ratio of 50:1) indicating that target cellswere directly lysed by activated T cells but not by soluble factors (datanot shown). T cells activated by KLH-pulsed DCs could lyse KLH-pulsed macrophages but not unpulsed macrophages regardless of thepresence or absence of soluble KLH during the CTL assays (Fig. 7b).In addition, CD81 and CD41 (partly) T cells activated by KLH-pulsed DCs could lyse KLH-pulsed macrophages but not macro-phages pulsed with a different antigen, ovalbumin (Fig. 7c). Cytolysis

was significantly blocked by anti-MHC class II moAb and partly byanti-CD4, anti-CD8, and anti-MHC class I (Fig. 7d). These resultsimply that KLH-pulsed DCs have induced specific CTLs that can lysetarget cells via cognate interaction.

Induction of Antileukemic CTLs by DCs Pulsed with IrradiatedAutologous Leukemic Cells in Vitro. On the basis of the abovefindings, we finally tried to induce specific CTLs against autolo-gous leukemic cells using DCs in three patients with AML. Bycoculturing with antigen-pulsed DCs for 7 days, autologous CD41

and CD81 cells proliferated significantly. They expanded prefer-entially in the vicinity of DC clusters (Fig. 2,G and H). As thenumber of DCs per well increased, proliferative responses of Tlymphocytes were elevated. When harvested at day 18, sensitizedT lymphocytes lysed autologous leukemic cells from all threepatients (Fig. 8,a–c). In patient A, the cytotoxicity of sensitized Tcells was specific for autologous leukemic cell (percentage cyto-toxicity, 62.88 6 8.25% at 50:1 E:T ratio), with no effect onallogeneic leukemic cell (percentage cytotoxicity, 4.536 1.25% at50:1 E:T ratio) or EBV-transformed lymphoblastoid cell lines(percentage cytotoxicity, 1.336 1.11% at 50:1 E:T ratio; Fig. 8d).The results imply that DCs developed from CD341 PBSCs pos-sessed the capacity to present leukemia antigens to autologous Tcells and to induce specific CTLs.

Induction of CTLs Is Dependent upon Cluster Formation byDCs. When the DC cluster formation was interrupted by frequentpipetting, specific CTL induction to KLH (Fig. 9) or to leukemia cells(Fig. 8d) was greatly retarded.

Fig. 5. Double-color flow cytometric analyses of CD1a1 cells generated from CD341 cell culture at day 11. Sixteen to 40% of CD1a1 cells coexpressed HLA-DR, CD40, and CD83.

Fig. 6. Immunostimulatory capacity of developed DCs in a primary allogeneic MLR.Note an intense alloantigen-presenting capacity of DCs (m) compared to PBSCs (F) ormononuclear cells (E).

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DISCUSSION

Here, we could generate DC clusters containing phagocytic cellsfrom CD341 cell fraction of human PBSCs. The developed cells havebeen proven to be of DC lineage by their characteristic phenotype andstrong stimulating capacity in primary allogeneic MLR (19). AlthoughPBSC fraction-derived CD341 HPCs were relatively less pure afterpositive selection and expanded to a lesser degree than bone marrow-derived HPCs, PBSC-derived HPCs produced more DCs, suggestingthat they contained more committed DC precursor cells (20). Cur-rently, many laboratories develop DCs from peripheral blood CD141

cells. However, it is not easy to obtain large number of DCs enoughfor immunotherapy by this method because generated DCs usually donot expand and usually do not survive for long time (21). Therefore,we suggest that PBSCs collected through leukapheresis after G-CSFtreatment are an efficient and economical source for developing alarge number of viable DCs from CD341 HPCs.

Phagocytic DCs in the DC clusters showed a relatively immaturephenotype. Immature DCs can take up antigens during a definiteperiod (8, 9), and in some conditions, they can avidly internalize largeparticulates including microorganisms (10–12) and apoptotic cells(22). For DCs to obtain antigen-presenting ability, two sequential

events may be necessary: (a) capturing and processing of particulateantigens by immature DCs together with active biosynthesis of abun-dant MHC class II molecules and invariant chain (23, 24) and numer-ous acidic endocytic vacuoles (25); and (b) antigen presentation bymature DCs through enhanced accessory or immunostimulatory func-tions, namely, adjuvant effects (7). An example of this is that seen inthe handling of soluble proteins (23, 24) and particles (12) by epider-mal Langerhans cells. In fact, isolated DCs from human peripheralblood, which mainly consisted of DCs without phagocytic activity(26), could not induce CTLs to KLH unless antigen was directlyintroduced by osmotic lysis (14). Therefore, phagocytic DC may becritical in our study for CTL induction to leukemic cells as well asprotein antigens, possibly through phagocytosing them before proc-essing.

The DC clusters could present an exogenous protein antigen, KLH,to not only CD41 but also CD81 T lymphocytes, which, in turn,acquire the ability to proliferate and kill the target KLH-pulsedmacrophages through antigen-specific cognate interactions. It wasgenerally believed that exogenous antigens were handled by endocyticpathway, loaded on MHC class II but not on class I molecules ofAPCs, and were recognized only by CD41 T lymphocytes (27, 28).

Fig. 7. Specificity of anti-KLH CTL induction by developed DCs. A representative result of two experiments.a, autologous T cells activated by KLH-pulsed DCs (F) but not byKLH-pulsed macrophages (m) lysed target macrophages.b, T cells activated by KLH-pulsed DCs could lyse KLH-pulsed macrophages (E) but not unpulsed macrophages, regardlessof a presence (F) or absence (M) of soluble KLH during CTL assay.c, both CD41 and CD81 T cells activated by KLH-pulsed DCs could lyse KLH-labeled macrophages (MandF) but not macrophages labeled with different antigen, ovalbumin (OVA-Mf, ✖ and ‚). d, blocking of CTL activity with moAb. Specific cytolysis by activated T cells (F) wassignificantly blocked by anti-MHC class II moAb (M) and partly by anti-CD4 (Œ), anti-CD8 (m), and anti-MHC class I (E).

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However, when exogenous antigens are internalized into phagosomesof APCs, they can be loaded on MHC class I molecules (29, 30), thusstimulating CD81 T cells as well. Therefore, we suggest that phago-cytic activity of the DC clusters may be essential for processingexogenous antigens for binding to MHC class I as well as class IImolecules by which both CD81 and CD41 CTLs can be generated.Because immunogenic peptides loaded on MHC molecules are longlived on the surface of DCs, compared to those on macrophages andB cells, T cells may be stimulated by DCs even if they are added 4days after antigen pulse to the DC culture.

We could induce autologous antileukemic CTLs in three AMLpatients using the DC clusters pulsed with irradiated leukemic cells. Itmay be important that leukemic cells receive irradiation, which leadscells to commit apoptosis because DCs are suggested to induce CTLsby acquiring antigens from apoptotic cells but not necrotic cells (22).Once pulsed during immature phase, mature DCs may be able toactivate both CD81 and CD41 T lymphocytes against tumor cells ina similar fashion as KLH antigens. Although most of other laborato-ries use leukemia-derived peptides as antigens, it is not always easy toobtain antigenic peptides from different types of leukemia patients.This study should overcome this problem because our DC clusters canbe pulsed with a whole leukemia cell body. Similarly, we could

induce allogeneic CTLs to a leukemic cell line using cord bloodCD341 cell-derived DCs in a preliminary study (18). Therefore, wesuggest that manipulation of the immunogenic potential of PBSCs toproduce antileukemic effect in autologous PBSCT (1, 3) is quitefeasible.

The DC clusters contained DCs of different maturational stagesbecause we did not enrich mature DCs. Because the mechanicaldisruption of the clusters abrogated CTL induction to hemocyanin orleukemic cells, these DC clusters seem to be the most essential fordeveloping T-cell responses against target cells. The stromal cell-likecells forming the foundation of the clusters may efficiently supportdevelopment of DC for antigen uptake and presentation. In thisrespect, putative endothelial cell progenitors were recently isolatedfrom adult human peripheral blood CD341 cells (31), and fibroblast-like cells with endothelial phenotype derived from CD341 cells ofumbilical cord blood are reported to support hematopoiesis (32).Similarly, it seems to be essential for T cells to make cell clusters(rosettes) with DC for effective induction of committed CD41 andCD81 T cells. DCs form rosettes with antigen-specific T cells, cre-ating a microenvironment in which immunity can develop (33, 34).

In conclusion, this study has demonstrated the successful inductionof antileukemic CTLsin vitro using an autologous DC culture. We

Fig. 8. Induction of anti-leukemic CTLs by DCs pulsed with irradiated autologous AML cells. In general, a mixture of CD81 and CD41 cells (F) showed stronger CTL activitythan CD81 (E) or CD41 (M) alone in patients A (a), B (b), and C (c). In patient A (d), cytotoxicity of sensitized T cells were specific to autologous leukemic cell (F) with no effecton allogeneic leukemic cell (✖) and EBV-transformed lymphoblastoid cell lines (M). When cluster formation was interrupted by frequent pipetting (Œ), specific CTL induction toautologous AML cells was greatly decreased.

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suggest that, to develop the phagocytic DC clusters from CD341 cellsmay be very critical for efficient CTL induction, not onlyin vitro butalso in vivo, in case of the leukemia immunotherapy together withautologous PBSCT. The study on the interrelationship between DCand T lymphocytes will become more and more important for activeimmunotherapyin vivo.

ACKNOWLEDGMENTS

We are grateful to Dr. Jonathan M. Austyn for critically reviewing themanuscript and for helpful discussions. Dr. Ralph M. Steinman is also ac-knowledged for his valuable suggestions.

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Fig. 9. Effect of the DC cluster disruption on CTL induction. When cluster formationwas interrupted by frequent pipetting (‚), specific CTL induction to KLH was greatlydecreased compared to the ordinary culture (F).

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1999;59:2150-2158. Cancer Res Shin-ichiro Fujii, Koji Fujimoto, Kanako Shimizu, et al. Leukemia PatientsLymphocytes against Leukemic Cells in Acute MyelogenousProgenitor Cells: Induction of Autologous Cytotoxic T

Hematopoietic+Clusters Generated from Human CD34Presentation of Tumor Antigens by Phagocytic Dendritic Cell

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