Leukemia-Derived Immature Dendritic Cells Differentiate into Functionally Competent Mature Dendritic Cells That Efficiently Stimulate T Cell Responses

Leukemia-Derived Immature Dendritic Cells Differentiate intoFunctionally Competent Mature Dendritic Cells ThatEfficiently Stimulate T Cell Responses1

Alessandro Cignetti,2* Antonella Vallario,*† Ilaria Roato,* Paola Circosta,*Bernardino Allione,§ Laura Casorzo,‡ Paolo Ghia,*† and Federico Caligaris-Cappio3*†

Primary acute myeloid leukemia cells can be induced to differentiate into dendritic cells (DC). In the presence of GM-CSF, TNF-�,and/or IL-4, leukemia-derived DC are obtained that display features of immature DC (i-DC). The aim of this study was todetermine whether i-DC of leukemic origin could be further differentiated into mature DC (m-DC) and to evaluate the possibilitythat leukemic m-DC could be effective in vivo as a tumor vaccine. Using CD40L as maturating agent, we show that leukemic i-DCcan differentiate into cells that fulfill the phenotypic criteria of m-DC and, compared with normal counterparts, are functionallycompetent in vitro in terms of: 1) production of cytokines that support T cell activation and proliferation and drive Th1 polar-ization; 2) generation of autologous CD8� CTLs and CD4� T cells that are MHC-restricted and leukemia-specific; 3) migrationfrom tissues to lymph nodes; 4) amplification of Ag presentation by monocyte attraction; 5) attraction of naive/resting andactivated T cells. Irradiation of leukemic i-DC after CD40L stimulation did not affect their differentiating and functional capacity.Our data indicate that acute myeloid leukemia cells can fully differentiate into functionally competent m-DC and lay the groundfor testing their efficacy as a tumor vaccine. The Journal of Immunology, 2004, 173: 2855–2865.

R elapse is the main reason of treatment failure in acutemyeloid leukemia (AML).4 Though �80% of AML pa-tients achieve complete remission after conventional che-

motherapy, �50% invariably relapse (1), underscoring the need ofnovel treatment strategies (2). One such strategy entails the devel-opment of immunotherapy approaches designed to obtain AML-specific CTLs potentially able to eradicate or control minimal re-sidual disease (3).

Priming of effective CTL responses requires the presentation ofrelevant Ags by a professional APC (4). In this context, AMLprovides the unique opportunity to derive APC from leukemiccells themselves, combining the expression of all available leuke-mic Ags with the presence of the several accessory and costimu-latory signals which are necessary to prime naive T cells (5). Thesimplest way to derive professional APC from myeloid malignan-cies is to induce the differentiation of leukemic cells into dendritic

cells (DC) (6, 7), which can actually be obtained upon in vitroculture of primary AML cells in the presence of various cytokinecombinations (8–11). Leukemic DC have proven to be potentstimulators of allogeneic T lymphocytes in mixed leukemia-lym-phocyte reactions, and a few reports also suggest that autologousCTL can be generated in vitro by stimulation with leukemic DC(9, 11–14).

In chronic myeloid leukemia and in AML, leukemic DC havebeen initially obtained in vitro by stimulation with GM-CSF to-gether with IL-4 and/or TNF-� (8–11, 15–17). With the lattercytokine combination, the leukemia-derived DC do not acquire afully mature phenotype but rather an immature one, displaying lowor intermediate levels of costimulatory molecules and low to un-detectable levels of the DC maturation marker CD83 (8, 12, 13,18–24); they also produce low to undetectable amounts of IL-12,another hallmark of DC maturation (11, 25, 26). Recent studieshave suggested that normal immature DC (i-DC) or semimatureDC (induced by TNF-�) injected in humans might have an inhib-itory effect on T cell function (27). On the contrary, normal matureDC (m-DC) are the most potent APC for efficient T cell priming invivo (28, 29). So far, the stepwise differentiation of leukemic blaststo i-DC and then to m-DC has never been studied and character-ized from a preclinical point of view (26, 30).

If DC of leukemic origin are planned to be used in vivo as acancer vaccine, it becomes crucial to determine whether they candifferentiate to m-DC not only in terms of phenotype but, mostimportantly, of function. To this end, we have studied 20 AMLpatients analyzing whether leukemic i-DC could fully differentiateto m-DC in response to CD40L and whether leukemic m-DC werefunctionally competent in terms of: 1) production of cytokines thatsupport Ag-specific T cell activation, proliferation, and Th1 dif-ferentiation; 2) generation of autologous T cell effectors; 3) mi-gration from tissues to lymph nodes; 4) amplification of Ag pre-sentation by attraction and recruitment of monocytes and otheri-DC; 5) attraction of resting and activated T cells. We here show

*Laboratory of Cancer Immunology, Institute for Cancer Research and Treatment,†Department of Oncological Sciences, University of Torino Medical School, and‡Unit of Pathology, Institute for Cancer Research and Treatment, Candiolo, Italy; and§Division of Hematology, Azienda Ospedaliera Santissimi. Antonio e Biagio, Ales-sandria, Italy

Received for publication December 22, 2003. Accepted for publication June 4, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the “Associazione Italiana per la Ricerca sul Cancro”(AIRC), Milano, and by Ministero dell’Universita e Ricerca Scientifica (MIUR),Roma, Italy.2 Address correspondence and reprint requests to Dr. Alessandro Cignetti, Laboratoryof Cancer Immunology, Institute for Cancer Research and Treatment, Strada Provin-ciale 142, Candiolo (TO), 10060, Italy. E-mail address: [email protected] Current address: Universita Vita Salute San Raffaele, 20132 Milano, Italy.4 Abbreviations used in this paper: AML, acute myeloid leukemia; DC, dendritic cell;BM, bone marrow; PB, peripheral blood; i-DC, immature DC; m-DC, mature DC;FISH, fluorescence in situ hybridization; MLTC, mixed lymphocyte-tumor culture;MDC, macrophage-derived chemokine; TARC, thymus and activation-regulated che-mokine; IP-10, IFN-�-inducible protein 10; MIG, monokine induced by IFN-�;PARC, pulmonary and activation-regulated chemokine.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

that leukemic blasts could be induced to differentiate into cellsfulfilling the phenotypic and functional criteria of m-DC in 10 of20 cases. Irradiation of leukemic i-DC after CD40 stimulation didnot affect their differentiation to m-DC in terms of phenotype, cy-tokine/chemokine production, and migration properties. Thesedata lay the ground for testing the efficacy of leukemic m-DC as avaccine aimed at eradicating minimal residual disease in a relevantnumber of AML patients.

Materials and MethodsPatients

After informed consent, samples from either bone marrow (BM) or pe-ripheral blood (PB) were obtained at diagnosis from 20 AML patients. Insome cases, samples were also collected during morphological remission(�5% BM blasts) after chemotherapy. Table I shows the main clinical anddiagnostic laboratory data of all cases.

Generation of leukemic and normal DC

BM or PBMCs were isolated after centrifugation over a density gradient(Lymphoprep; Nycomed, Oslo, Norway) and used immediately or cryo-preserved. Fresh or thawed primary AML cells were seeded into 25 or 75cm2 flasks at 1 � 106/ml and cultured in serum-free medium (X-Vivo15;BioWhittaker, Walkersville, MD). In four selected cases, leukemic cellswere obtained by depleting PBMC with a mixture of magnetically labeledmAbs against CD3, CD19, CD16, and CD56 (Miltenyi Biotec, Auburn,CA), resulting in a purity of �98% leukemic cells (Table I). To generateleukemic i-DC, purified or unfractionated AML cells were stimulated withGM-CSF, IL-4 (both at 80 ng/ml), and TNF-� (10 ng/ml) (BioSource In-ternational, Camarillo, CA). Cytokines and fresh medium were added ev-ery 4–5 days.

Normal i-DC were obtained from purified CD14� cells using GM-CSFand IL-4 as described elsewhere (31). To generate leukemic and normalm-DC, cells from i-DC cultures were CD40 cross-linked using 300 ng/mlof a rCD40L-FLAG-tag fusion protein supplemented with 300 ng/ml“CD40L enhancer” (Alexis Biochemicals, San Diego, CA). After 24, 48, or72 h, depending on the assay, cells were used for functional analysis.

Flow cytometric analysis

Unmanipulated and cultured AML samples were stained with FITC, PE, orPECy5-conjugated mouse mAbs using standard multicolor methodology,and analyzed by flow cytometry (FACS). Blasts and DC were gated basedon side light scatter profile vs CD45, as previously described (31). MousemAbs to the following Ags were used: CD1a, CD33, CD34, CD80, CD86,

CD40, HLA class I, HLA-DR (BD Pharmingen, San Diego, CA), CD13,CD14 (BD Biosciences, San Jose, CA), and CD83 (Immunotech, West-brook, ME). Unconjugated mAb to CCR7 (IgM) was obtained from BDPharmingen. PE-conjugated secondary goat polyclonal Ab against murineIgM was purchased from Southern Biotechnology Associates (Birming-ham, AL). For other chemokine receptor mAbs, see Ref. 31. Isotype-matched mAbs were used as negative controls. Samples were analyzedwith a FACSCalibur cytometer (BD Biosciences).

Fluorescence in situ hybridization (FISH) analysis

Interphase FISH was performed on CD83� purified leukemic m-DC and onnaive leukemic blasts (10). Three patients were selected according to theircytogenetic abnormality and interphase nuclei were analyzed using theprobes of interest. For 7q� and 5q� deletion (patients GE and VA), dualcolor FISH was performed, using probes labeled either with spectrumgreen or spectrum orange. A centromeric probe for chromosome 7 (7p11.1-q11.1) and a probe for 7q31 were used for the 7q� deletion, while a probefor 5p15.2 and a probe for 5q33-34 were used for the 5q� deletion (VYSIS,Downers Grove, IL). For monosomy 7 (patient CA), only the centromericprobe for chromosome 7 was used. Fixation, denaturation, and hybridiza-tion were conducted following manufacturers’ instruction. At least 200nuclei were examined under fluorescence microscopy.

RNA preparation and RT-PCR

RNA was extracted from 1 to 2 � 106 cells using RNAzol (Biotecx Lab-oratories, Houston, TX) following the manufacturer’s instructions. cDNAwas prepared at 42°C using a reverse-transcription mix containing Super-script II (Invitrogen Life Technologies, Grand Island, NY). PCR amplifi-cation of cDNA samples was performed for 30 or 35 cycles. For chemo-kines and �-actin primer sequences, see Ref. 32. For each cytokine andchemokine, proper positive controls were used. All reactions were con-ducted in a PerkinElmer thermocycler (Foster City, CA).

Cytokine and chemokine production

Supernatants were obtained from cultures that, after washing, were seededat 2 � 106cells/ml and kept in serum-free medium for 24 or 72 h. Super-natants from i-DC were obtained after stimulation of i-DC cultures withGM-CSF, TNF-� and IL-4; supernatants from m-DC were obtained afterstimulation of i-DC cultures with CD40L. Cytokine and chemokine pro-duction was measured by ELISA using the following commercially avail-able kits: IL-12, IL-12 HS (high sensitivity), IL-15, IL-18, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine(TARC), MCP-1, MIP-1�, MIP-3�, RANTES (R&D Systems, Minneap-olis, MN), IFN-�-inducible protein 10 (IP-10) (CytImmune Sciences, Col-lege Park, MD), IL-6, IL-8, IL-10 (Bender MedSystems, Vienna, Austria),and IL-16 (Technogenetics, Milano, Italy).

Table I. Patients’ characteristics

Patient Sex FAB Sample % Blasts Cytogenetic

Differentiation% Positive

CD14BlastsCD86i-DC m-DC

GA M M0 PB 78 �7 No No �5 �5CER F M0 BM 81a t (7;8) Yes Yes �5 79FE M M1 PB 44 ND No No �5 �5BO F M1 PB 88 ND No No �5 �5CA F M1 BM,b PB 95a �4, �5, �7 Yes Yes �5 76IE F M1 BM,b PB 95 46 XX No No �5 �5BE F M2 PB 92 46 XX No No �5 �5DA M M2 BM 70 5q�, 7�, 8�, 22� Yes Yes �5 88PE F M2 PB 77 ND No No �5 �5RA M M2 PB 85 46 XY No No �5 �5GI F M2 PB 87 46 XX No No �5 NDZA F M4 PB 95 46 XX Yes Yes 20 19VA F M4 BM,b PB 86 5q� Yes Yes 46 53BI M M4 PB 77 ND Yes Yes 36 90GE M M4 BM 72a 7q�, 11q� Yes Yes 10 95RO M M4 PB 95 ND No No 23 55FE M M4 PB 68 ND No No 6 �5FR F M5 BM, PB 89 46 XX Yes Yes 81 77CR F M5 PB 87a 46 XX Yes Yes 80 92SU M M5 PB 94 46 XY Yes Yes 82 98

a Purified cells were used to generate leukemic DC.b BM was used to generate leukemic DC.

2856 AML BLASTS DIFFERENTIATE INTO FUNCTIONAL m-DC

Stimulatory function of the leukemic DC

Allogeneic mixed lymphocyte-tumor cultures (MLTC) were set up withPBMC from healthy donors as responders and naive leukemic cells, leu-kemic, and normal DC as irradiated stimulators, as previously described(10). All cultures were performed in serum-free medium (AIM-V; Invitro-gen Life Technologies). Normal DC were derived from CD14� purifiedPBMC of healthy sibling donors that were HLA-matched with the patient.Autologous MLTC were similarly performed, but responder T cells usedfor proliferation assay were previously cocultured with different stimula-tors, i.e., autologous blasts, i-DC or m-DC. Briefly, patients’ PBMC wereprimed with autologous blasts, i-DC, or m-DC and similarly restimulatedafter 7 days. Proliferation was then measured by pulsing cells with[3H]TdR 4 days after a last stimulation of all differently cultured T cellswith autologous blasts.

Induction of autologous T cell effectors

AML blasts were differentiated to leukemic m-DC, irradiated at 30 Gy andused to stimulate autologous PBMC obtained after complete remission.Autologous MLTC were set up with patients’ PBMC (107 nonadherentPBMC) cocultured with 2 � 106 leukemic m-DC in AIM-V serum-freemedium. On days 7, 14, and 21, PBMC were restimulated with autologousirradiated leukemic m-DC. rIL-2 (20 IU/ml) was added 72 h after thesecond stimulation and then every 3–4 days. At day 28, PBMC were sep-arated into CD4� and CD4� cells using CD4� microbeads (Miltenyi Bio-tec) and purified fractions were further restimulated with autologous leu-kemic m-DC. Assay for functional activity was performed 5–7 days afterthe fourth or fifth restimulation.

ELISPOT assay

The ELISPOT assay was performed using cells from MLTC as effectors(4 � 104cells/well) and the following autologous targets (1 � 104/well):naive leukemic blasts, CD14� purified monocytes, and EBV-transformedB cells. ELISPOT assays were conducted according to manufacturer’s in-struction (Mabtech, Stockholm, Sweden), with few modifications (33). Be-fore developing the assay, T cells were routinely transferred from theELISPOT plate to a normal 96-well plate and viability of recovered cells

was verified after 2 additional days of culture (without cytokines) bychecking all wells under the microscope and also by randomly countingsome wells with trypan blue exclusion. This allowed us to rule out cell lossor cell death due to technical inaccuracy and also to check correspondencebetween cell viability and IFN-� activity in individual wells. By perform-ing this control of cell viability, in fact, we could exclude that a remarkableT cell death had occurred in those same wells where IFN-� activity wasdetected. Blocking experiments were performed by preincubating targetcells for 20� with 25 �g/ml of an anti-HLA class I (clone W6.32) or ananti-HLA class II (clone L243) and an isotype-matched irrelevant mAb (allfrom BD Pharmingen). Spots were counted by a computer-assisted ELIS-POT reader (AID; Bioline, Torino, Italy). Indicated spot numbers perseeded PBMC represent mean values of duplicates.

Chromium release assay

Target cells (primary AML cells) were incubated with 50 �Ci ofNa2

51CrO4 (Amersham Biosciences, Cologno Monzese, Italy) for 2 h at37°C and washed. Thereafter, 5 � 103 viable target cells were incubatedwith CD8� effector cells from MLTC at different E:T ratios for 4 h at 37°C.After incubation, the supernatants were collected and radioactivity wasmeasured on a gamma counter. Spontaneous release was determined byharvesting the supernatant of target cells incubated in the absence of ef-fector cells and maximum release was determined by resuspending targetcells incubated in the absence of effector cells. The percent of specific lysiswas calculated as follows: (experimental release � spontaneous release/maximum release � spontaneous release) � 100%. The spontaneous re-lease always ranged between 5 and 20% of the total label incorporated bythe cells. To determine class I restriction in the recognition of target cells,blocking studies were performed by adding 25 �g/ml anti-HLA class ImAb (clone W6.32) to 51Cr-labeled targets 30 min before the assay. An Abof the same isotype was used as negative control. The Abs were not washedout before mixing target and effector cells.

Generation of T cell subsets and migration assay

CD4� and CD8� T cells were purified from PBMC by depleting CD19�,CD56�, CD16�, CD14�, and CD8� or CD4� cells with magnetic mi-crobeads (Miltenyi Biotec), and activated by stimulation with anti-CD3 and

FIGURE 1. Maturation of leukemic DC up-modulates CD83 and CD80 expression. Blasts, leukemic, and normal i-DC and m-DC cultures were analyzedby flow cytometry for the expression of CD83 (A) and CD80 (B). Data are expressed as percent of positive cells and each line represents data obtainedwith leukemic (n � 10) or normal CD14� (n � 8) samples.

2857The Journal of Immunology

anti-CD28 (100 ng/ml and 1 �g/ml, respectively) (BD Pharmingen). Purityof the CD4� or CD8� fraction was always �95%. Activated T cells werecultured with IL-2 (2 ng/ml; Chiron, Emeryville, CA) and IL-7 (5 ng/ml;R&D Systems) in the presence of irradiated allogeneic feeder cells(PBMC) for 7 to 14 days. Naive/resting CD4� and CD8� T cells wereprepared from nonadherent PBMC negatively depleted of CD14�, CD19�,CD56�, CD45RO�, CD25�, HLA-DR�, and CD8� or CD4� cells usinggoat anti-mouse Ig-coated magnetic microbeads (Miltenyi Biotec). Purityof the resulting CD45RA�/CD4� or CD8� cells was always �95% ascontrolled by FACS.

Chemotaxis assays were performed using the Transwell system (5 �mpores; Costar, Cambridge, MA), as previously described (31, 32). For che-motaxis of leukemic m-DC, escalating doses of rMIP-3� (R&D Systems)were added to X-Vivo15 and distributed on the lower compartment of thechemotaxis system; CCR7� leukemic m-DC were placed into the Trans-well inserts. Plates were incubated for 4 h. A control condition with norecombinant chemokine was also included. For normal monocytes and Tcell subset migration, supernatants were collected from leukemic and nor-mal DC cultures and used at 100% v/v or with serial 1/2 dilutions anddistributed on the lower compartment of the chemotaxis system. Mono-cytes or T cells were then placed into the Transwell inserts. T cells wereobtained as described above, while normal monocytes were obtained fromhealthy donors with the monocyte isolation kit as described for DC gen-eration. In selected cases, monocytes or T cells were incubated before the

migration assay with saturating concentrations of MCP-1 (2 �g/ml), MDC,or IP-10 (4 �g/ml) for 30 min to down-modulate CCR2, CCR4, andCXCR3 expression, respectively, as evaluated by FACS analysis. Plateswere incubated for 2, 4, and 12 h (activated T cells, monocytes, and naiveT cells, respectively). A negative control condition with fresh medium wasalways included.

In all migration assays, the liquid accumulated in the lower compart-ment of the chemotaxis system was carefully recovered and migrated cellswere counted by flow cytometry. Results are expressed as the percentageof migrating cells, i.e., number of migrated cells/number of inputcells � 100.

Statistical analysis

The statistical significance ( p values) between i-DC and m-DC or betweenleukemic and normal i-DC and m-DC was analyzed by two-tailed Studentt test for paired data.

ResultsGeneration of leukemic and normal i-DC and m-DC

We analyzed 20 AML samples to determine whether AML blastscould be induced to differentiate first to i-DC and then to m-DC.When a significant proportion of cells cultured in the presence ofGM-CSF, TNF-�, and IL-4 started to show evidence of DC mor-phology, their phenotype was analyzed by flow cytometry (usually

FIGURE 2. Maturation of leukemic DC induces IL-12 and IL-15 pro-duction and down-modulates IL-10 production. A, Supernatants from i-DCand m-DC cultures were analyzed for IL-12p70 production by ELISA.Cells were seeded at 2 � 106 cells/ml and harvested after 24 h. Datarepresent the mean � SD of 10 leukemic samples and of 4 normal samples(CD14-derived DC). �, p � 0.01; §, p � 0.05. B, Supernatants from blasts,i-DC, and m-DC cultures were analyzed for IL-15 and IL-10 production byELISA. Cells were seeded at 2 � 106 cells/ml and harvested after 72 h.Data represent the mean � SD of 10 leukemic samples. For blasts andi-DC, IL-10 samples were divided in two subgroups, according to theirdifferent pattern of IL-10 production. �, p � 0.01; §, p � 0.05.

FIGURE 3. Leukemic m-DC are better T cell stimulators than i-DC inallogeneic and autologous MLTC. A, Allogeneic MLTC was performedwith 105 allogeneic PBMCs as responder cells and different numbers ofthawed AML blasts or leukemic and normal i-DC and m-DC as stimulatorcells at responder/stimulator ratios (R/S) ranging from 3:1 to 30:1. Figureshows data obtained at a R/S of 30:1. B, Autologous MLTC was performedsimilarly to allogeneic MLTC, but responder cells were patients’ T cellsthat had been differently stimulated at week intervals with autologousblasts, i-DC, or m-DC. Data in A and B are presented as mean � SE ofthree cases in each panel for a total of six patients analyzed. �, p � 0.001between i-DC and m-DC groups (both leukemic and normal) analyzed witha t test for paired data. All cultures were conducted in serum-free medium.


FIGURE 4. Leukemic m-DC generateMHC-restricted CD8� and CD4� T cells in au-tologous MLTC. CD4� (A) and CD8� (B andC) T cells from autologous MLTC were testedfor their activity against naive autologous blasts,monocytes and EBV-transformed B cells. A andB, T cell reactivity was quantified using anELISPOT assay for IFN-�. Results shown arereferred to a T cell-target ratio of 4:1, i.e., 4 �104 CD4� or CD8� T cells and 1 � 104 blasts,monocytes, or EBV-transformed B cells. Figureshows mean � SE of four experiments forCD4� T cells and three experiments for CD8�

T cells. C, CTL activity was determined by 51Crrelease assay. Figure shows mean � SE of twoindependent experiments.


after 7–14 days) (10). As CD1a and CD83 are considered markersof i-DC and m-DC, respectively, (34, 35), AML cells were con-sidered to have differentiated into i-DC when �50% of cells werepositive for CD1a and/or CD80. Samples were then stimulatedwith CD40L and analyzed for CD83 expression after 24, 48, and72 h of culture. When at least 50% of the CD80� cells coexpressedCD83, cultured AML cells were considered to have become leu-

kemic m-DC. According to these criteria, AML blasts were able todifferentiate to m-DC in 10 of 20 cases (see Table I). Before dif-ferentiation, all 10 cases were CD86-positive, while only 8 of 10cases were CD14-positive. On the contrary, cells were CD86 andCD14 positive only in 1 of 10 cases in which DC differentiationwas not achieved (Table I). Fig. 1 shows how the up-modulation ofCD83 and CD80 is associated with maturation of leukemic as wellas of normal DC. The difference in CD83 and CD80 expressionbetween i-DC and m-DC (both leukemic and normal) is statisti-cally significant, when data are analyzed with a t test for paireddata ( p � 0.05 and p � 0.005 for CD80 and CD83, respectively).We also analyzed the expression of CD86, CD40, HLA class I, andHLA-DR in leukemic i-DC and m-DC, without finding a signifi-cant difference between the two subsets (data not shown).

To prove that CD40L-induced maturation does not cause en-richment of residual normal cells over the original leukemic cells,we performed cytogenetic FISH analysis in three selected cases.Cells were analyzed for the presence of 7q� (patient GE) and 5q�

(patient VA) deletion or for monosomy 7 (patient CA). FISH anal-ysis revealed that, before culture, 89 and 76% of unfractionatedblasts were positive for 7q� and 5q� deletion, respectively, and72% of blasts were positive for monosomy 7. Following culture,79, 70, and 73% of CD83� purified cells displayed the same cy-togenetic abnormality, respectively (not shown). These data pro-vide proof of principle that the m-DC obtained in our culture con-ditions are of leukemic origin.

Maturation of leukemic DC induces the production of IL-12 andIL-15 and down-modulates the production of IL-10

To further evaluate the differentiation stage of leukemic DC, wemeasured by ELISA the production of IL-12p70 by leukemic i-DCand m-DC and compared it to CD14-derived normal counterparts.As shown in Fig. 2A, supernatants from leukemic and normal i-DCcultures showed negligible IL-12 production. On the contrary, su-pernatants from both normal and leukemic m-DC cultures showedsignificant amounts of IL-12 production. Similarly to IL-12, alsolevels of IL-15 production in both leukemic and normal DC sub-populations were significantly higher in m-DC than in i-DC (Fig.2B and not shown).

As for IL-10, we found two different patterns of production (Fig.2B): 1) IL-10 was produced by leukemic blasts but not by leuke-mic i-DC and m-DC in 5 of 10 cases; 2) IL-10 was not secreted byleukemic blasts but its production was induced in leukemic i-DCand down-regulated in leukemic m-DC in the remaining 5 of 10cases. Altogether, the data indicate that IL-10 can be expressed andproduced by leukemic blasts or leukemic blast-derived i-DC, butnot by leukemic blast-derived m-DC.

FIGURE 5. Mature leukemic DC express CCR7 and migrate in re-sponse to MIP-3�. A, Blasts and leukemic i-DC and m-DC cultures wereanalyzed by flow cytometry for the expression of CCR7. A value of p ofthe m-DC group compared with the i-DC group is �0.01 when analyzedwith a t test for paired data. B, Migration assay of CCR7� leukemic m-DCin response to rMIP-3�. Migration of four leukemic and three normalm-DC independent cases pooled together are presented.

Table II. Chemokine expression by leukemic and normal DC

DC Type

Chemokines

MCP-1 MDC TARC MIP-1� MIP-3�

PCRa ELISAb PCRa ELISAb PCRa ELISAb PCRa ELISAb PCRa ELISAb

Leukemici-DC 10/10 1,451 (142–2,378) 10/10 6,610 (719–8,661) 10/10 6,732 (3,154–9,576) 10/10 609 (13–3,884) 5/9 7 (0–72)m-DC 10/10 750 (585–1,829) 10/10 7,859 (352–8,573) 10/10 7,588 (3,559–12,145) 10/10 181 (152–1,201) 7/9 8 (0–102)

CD14i-DC 4/4 1,419 (648–2,411) 4/4 8,731 (1,451–9,145) 4/4 7,242 (273–1,884) 4/4 2,006 (415–3,597) 4/4 0m-DC 4/4 995 (304–1,546) 4/4 8,968 (8,849–9,929) 4/4 9,957 (3,709–12,024) 4/4 ND 4/4 19 (0–204)

a Number of positive samples per total tested.b Median (range) of picograms per milliliter.


Leukemic m-DC stimulate T cell proliferation better than i-DCin allogeneic and autologous MLTC

To prove that leukemic m-DC are capable of supporting T cellactivation and proliferation better than i-DC counterparts, we in-vestigated their ability to stimulate allogeneic T cells in primaryMLTC. In three cases analyzed, we found that m-DC elicited aproliferation significantly higher than i-DC and that the stimula-tory activity of leukemic i-DC and m-DC is superimposable to thatof normal counterparts. CD14-derived normal i-DC and m-DCwere obtained from HLA-matched sibling donors (Fig. 3A). Wealso compared the stimulatory activity of leukemic m-DC with thatof i-DC in tertiary MLTC in three additional cases. Patients’PBMC were repeatedly stimulated with autologous blasts, i-DC, orm-DC and their proliferation in response to naive autologous blastswas measured after 3 wk. Again, we found that m-DC elicitedsignificantly higher proliferation of autologous T cells than i-DCcounterparts (Fig. 3B).

Leukemic m-DC generate autologous T cell effectors that areMHC-restricted and leukemia-specific

PBMC from patients were repeatedly stimulated in vitro by autol-ogous leukemic m-DC and analyzed for their ability to recognizenaive leukemic blasts. Cells were purified according to theirCD4 and CD8 expression (�94% purity after selection), restimu-lated once more with leukemic m-DC and tested after 7 days byELISPOT. We found that stimulation with leukemic m-DC gen-erates CD8� and CD4� T cells that release IFN-� (Fig. 4, A andB) in response to naive leukemia blasts in three of four and four offour cases, respectively. In one AML patient, the initial number ofCD8� cells was very low and the final yield of CD8� T cells wasnot enough to perform functional assays. CD4� T cells releasedIFN-� but not IL-4 in response to naive leukemic blasts (notshown). MHC restriction of CD8� and CD4� effectors was provedby the inhibition of IFN-� release observed with specific anticlass-I or class-II mAb, respectively, but not with an irrelevantmAb of the same isotype (IgG2a). Leukemia specificity was fur-ther confirmed by the lack of recognition of autologous myeloid(PB monocytes) and lymphoid (EBV-transformed B cells) targets(Fig. 4, A and B). Finally, we also determined whether CD8� Tcells from MLTC could mediate effective killing of unmanipulatedblasts in a traditional cytotoxicity assay. In two of two cases an-alyzed, CD8� T cells efficiently lysed both autologous blasts andleukemic m-DC (Fig. 4C). Killing of leukemic m-DC was signif-icantly higher than that of primary blasts. Lysis of AML blasts wasblocked by a mAb to HLA-class I, but not by an irrelevant, iso-type-matched, control mAb (Fig. 4C).

Leukemic m-DC but not i-DC express CCR7 and migrate inresponse to MIP-3�

Maturing DC up-regulate CCR7 expression to acquire the abilityto migrate from inflamed tissue to draining lymph nodes in re-

sponse to CCR7 ligands (MIP-3�, secondary lymphoid tissue che-mokine). CCR7 was not expressed by leukemic i-DC while a sta-tistically significant induction of CCR7 expression was observedin leukemic m-DC (Fig. 5A). No significant difference in CCR7expression was observed between leukemic and normal m-DC (notshown). To prove that leukemic m-DC can effectively migratethrough CCR7 engagement, we performed a chemotactic assay invitro, where leukemic m-DC were exposed to rMIP-3�. In allfour samples analyzed, leukemic m-DC migrated in response toMIP-3�, similarly to normal m-DC (Fig. 5B).

Leukemic i-DC and m-DC produce inflammatory chemokinesthat induce the migration of PB monocytes

To amplify Ag presentation, normal DC also produce chemokinesto attract monocytes and other DC to inflamed tissue and to sec-ondary lymphoid organs. We analyzed the expression of MCP-1,MIP-1�, RANTES, IL-8, MIP-1�, and MCP-4 by RT-PCR in bothleukemic and normal DC subsets. mRNA for all these chemokineswas detected in the vast majority of samples (Table II). Amongthese chemokines, we analyzed by ELISA the actual production ofMCP-1 (Fig. 6A), MIP-1�, and IL-8 (Table II) and found that theyare also released at significant levels by both leukemic i-DC andm-DC. We next tested whether supernatants from DC culturescould attract in vitro normal monocytes in a chemotactic assay.Fig. 6B shows that supernatants from both leukemic i-DC andm-DC similarly induce the migration of freshly isolated PBmonocytes.

Considering the higher levels of CCR2 expression in normalmonocytes compared with the other chemokine receptors analyzed(not shown), we postulated that their migration in response to leu-kemic DC supernatants was mainly CCR2 mediated. We per-formed “desensitization” experiments where, before chemotacticassay, CCR2 expression was down-regulated by incubating targetcells with high doses of rMCP-1. CCR2 desensitization induced asignificant inhibition of monocyte migration in response to leuke-mic DC supernatants close to that obtained with rMCP-1 alone(Fig. 6C).

Leukemic i-DC and m-DC produce chemokines that induce themigration of naive and activated T cells

Normal DC also produce chemokines that attract T cells to thesites of Ag presentation. In particular, MDC/TARC and IP-10/monokine induced by IFN-� (MIG) are known to promote theinteraction of DC with recently activated T cells that respectivelyexpress CCR4 and CXCR3. We found that both i-DC and m-DCof leukemic origin produce considerable amounts of MDC andTARC, similarly to CD14-derived normal DC (Fig. 7A and TableII). A trend toward a higher level of MDC and TARC productionby m-DC than by i-DC was observed, which did not reach statis-tical significance. We also found that, in most cases, leukemic and

Table II. Continued

Chemokines

IP-10 RANTES IL-8 IL-16 MIP-1� MCP-4 MIP-3� PARC MIG

PCRa ELISAb PCRa ELISAb PCRa ELISAb PCRa ELISAb PCRa PCRa PCRa PCRa PCRa

6/10 35 (0–73) 10/10 29 (0–633) 10/10 1468 (144–2,485) 10/10 973 (504–1,886) 10/10 10/10 5/10 3/10 6/107/10 50 (0–674) 10/10 ND 10/10 ND 10/10 1,182 (431–2,399) 10/10 10/10 10/10 5/10 7/102/4 47 (0–257) 4/4 33 (7–215) 4/4 2,086 (255–2,464) 4/4 642 (285–1,203) 4/4 4/4 2/4 3/4 3/43/4 27 (0–110) ND ND ND ND 4/4 409 (375–1,658) ND 4/4 4/4 4/4 4/4


FIGURE 6. Leukemic i-DC and m-DC produce inflammatory chemo-kines that induce the migration of PB monocytes. A, Leukemic i-DC andm-DC produce MCP-1. Supernatants from blasts, i-DC, and m-DC cultureswere analyzed for MCP-1 production by ELISA. Cells were seeded at 2 �106 cells/ml and harvested after 72 h. Data represent the mean � SD of 10leukemic samples and of 4 normal samples (CD14-derived DC). B, Super-natants from leukemic DC cultures induce migration of PB monocytes.Chemotaxis of PB monocyte cells in response to leukemic and normali-DC and m-DC supernatants. Fresh PB monocytes were isolated by de-pleting T, B, and NK cells from PBMC. Data represent mean � SD of fourleukemic i-DC and m-DC samples and of three normal i-DC and m-DCsamples (CD14-derived DC). C, Chemotaxis of PB monocytes in responseto leukemic i-DC (n � 3) and m-DC-derived supernatants (n � 3) orrMCP-1, before (f) or after (u) CCR2 down-regulation by receptor de-sensitization with high dose rMCP-1 (2 �g/ml). The difference in migrationobtained before and after desensitization is statistically significant in allthree groups (p � 0.01).

FIGURE 7. Leukemic i-DC and m-DC produce lymphoid chemokines thatinduce the migration of resting and activated CD4� and CD8� T cells. A,Supernatants from blasts, i-DC, and m-DC cultures were analyzed for MDCproduction by ELISA. Cells were seeded at 2 � 106 cells/ml and harvestedafter 72 h. Data represent the mean � SD of 10 leukemic samples and of 4normal samples (CD14-derived DC). B, Chemotaxis of resting/naive and ac-tivated CD8� T cells in response to leukemic and normal i-DC and m-DCsupernatants. Purified CD8�/CD45RA� T cells were obtained from freshPBMC by negative selection. Activated T cells were obtained by stimulationof purified CD8� cells with anti-CD3 and anti-CD28 (see Materials and Meth-ods). Five independent experiments are pooled together. Data representmean � SD of four leukemic i-DC and m-DC samples and of three normali-DC and m-DC samples (CD14-derived DC). The percent of cells migratingin the presence of medium alone was subtracted from each data point. C,Chemotaxis of CD8�-activated lymphocytes in response to leukemic i-DC andm-DC-derived supernatants (n � 3) or rIP-10 and rMDC, before and afterCXCR3 or CCR4 down-regulation by receptor desensitization with rIP-10 (4�g/ml) and MDC (4 �g/ml), respectively. Three independent sets of experi-ments are pooled together. The percent of cells migrating in the presence ofmedium alone was subtracted from each data point. The difference in migra-tion obtained before and after desensitization is statistically significant in allthree groups (p � 0.05).


normal DC subsets express mRNA for IP-10 and/or MIG. As doc-umented by ELISA, IP-10 was detected at the protein level as well,without a significant difference between leukemic i-DC and m-DC.

Finally, also MIP-3� and pulmonary and activation-regulatedchemokine (PARC) were expressed at the mRNA level by leuke-mic and normal DC subsets (Table II). Only MIP-3� expressionwas significantly up-regulated in m-DC as compared with i-DC.MIP-3� and PARC are known to mediate the attraction of naive/resting T cells to the T cell area of secondary lymphoid organs. Toprove that the chemokines expressed and/or released by leukemicDC can attract naive/resting T cells, we purified CD8�/CD45RA�

cells from fresh PBMC and tested them in a chemotactic assay.CD8�/CD45RA� T cells migrated in response to both i-DC andm-DC supernatants, without significant differences between thetwo subsets (Fig. 7B). We also found that supernatants from leu-kemic i-DC and m-DC were equally capable of inducing migrationof activated CD8� T lymphocytes (Fig. 7B). Migration of naive/resting and activated CD4� cells in response to leukemic DC su-pernatants was slightly (but not significantly) higher than that ofCD8� counterparts (not shown), possibly due to the additionaleffect of IL-16 on CD4� cells (36). In fact, IL-16 was detected byELISA in the leukemic DC supernatants (Table II). Regardless ofthe T cell subtype analyzed, the levels of migration induced byleukemic DC supernatants were not different from those inducedby normal DC supernatants (Fig. 7B).

The analysis of the chemokine secretion pattern suggests that,among the several chemokines detected at the protein level in leu-kemic DC supernatants, at least two chemokine/chemokine recep-tor pairs might be involved in the attraction of activated T cells:IP-10-MIG/CXCR3 and MDC-TARC/CCR4. To investigate thisissue, we performed again desensitization experiments as de-scribed in the previous paragraph. We found that CXCR3- andCCR4-desensitized T cells migrated significantly less than normalcounterpart cells in response to leukemic DC supernatants.(Fig. 7C).

Irradiation of leukemic i-DC after CD40L stimulation does notaffect their differentiation to m-DC

To be used as a vaccine in vivo, leukemic DC should be irradiatedto prevent proliferation of residual leukemic cells. To evaluate howirradiation affects maturation and function of leukemic DC, westimulated i-DC cultures from three different patients with CD40Land after 4 h we exposed them to escalating doses of gamma rays.The following parameters were then assessed at different timepoints: viability, expression of maturation markers, cytokine/che-mokine production, and chemotactic activity. As shown in Fig. 8,a progressive decrease in cell viability was observed, which wasmore pronounced after the third day of treatment, and completecell death was seen at day 4. Despite cell death, remaining livingcells showed induction of CD83 and CCR7 expression similar to

FIGURE 8. Irradiation of leukemic i-DC after CD40 stimulation doesnot affect their differentiation to m-DC. i-DC cultures were stimulated withCD40L (day 0) and, after 4 h, were gamma-irradiated with three differentdoses (10, 30, and 60 Gy). Irradiated and nonirradiated cells were thenanalyzed at different time points as indicated. A, Viability as determined bycounting cells on a Bauer chamber after staining with a 0.4% trypan bluesolution to exclude dead cells; B and C, phenotype as determined by FACSanalysis of residual living cells (gate on living DC was set as described inMaterials and Methods); D, migration in response to rMIP-3� (200 ng/ml);E, chemokine and cytokine production as determined by testing culturesupernatants harvested 24 h after irradiation. All data are referred to onerepresentative case of three analyzed.


nonirradiated cultures, which was maintained for up to 60 h. More-over, supernatants harvested 24 h after irradiation showed no dif-ference between treated and untreated cells in terms of chemokineand cytokine production. Fig. 8 shows ELISA data for TARC andIL-12; similar data were obtained for IL-15, IL-10, MDC, andMCP-1. Finally, irradiated CCR7� m-DC were able to migrate inresponse MIP-3� and levels of migration were similar betweenirradiated and nonirradiated cells up to 48 h. Thus, irradiated leu-kemic DC maintain their functional properties up to at least 2 daysafter treatment.

DiscussionThe aim of this study was to provide evidence that DC of leukemicorigin may be used in vivo as a tumor vaccine following inductionof maturation. For clinical efficacy, leukemic DC should not onlypresent endogenous tumor Ags to T cells, but also carry out severalactivities that normal myeloid DC have in vivo, such as migrationfrom tissues to draining lymph nodes, attraction and recruitment ofnew APC and T cells, and induction and sustenance of T cellactivation and proliferation. They should induce a Th1 rather thana Th2 or T regulatory type of T cell response (27, 37) and, moreimportantly, generate a leukemia-specific T cell response.

In this study, we show that leukemic i-DC can be induced tofully differentiate into m-DC using CD40L as maturating agent,and that leukemic m-DC fulfill the functional criteria listed above.For instance, leukemic m-DC (but not i-DC) up-regulate CCR7expression and are able to migrate in vitro in response to the CCR7ligand MIP-3�. Moreover, leukemic m-DC (but not i-DC) secreteIL-12 and IL-15, two cytokines that support T cell activation, pro-liferation, and differentiation, and down-regulate the secretion ofIL-10, which has inhibitory activity on T cells and on DC as well.This pattern of cytokine production finds functional correspon-dence in the superior capacity of leukemic m-DC to stimulate al-logeneic as well as autologous T cell proliferation, when comparedwith i-DC. More importantly, the ability of m-DC to generate bothCD8� and CD4� leukemia-specific T cells was also documentedin the autologous setting. Other groups have shown that leukemia-derived DC (that were obtained otherwise than inducing matura-tion of i-DC with CD40L) can stimulate T cell responses in vitro(9, 11–14). However, this is the first study that provides concurrentevidence that the antileukemic activity is MHC restricted, is di-rected against unmanipulated blasts, resides specifically in purifiedCD8� or CD4� subsets, and can be obtained using serum-freemedium, which is mandatory for clinical purposes. In addition, wehave shown that normal myeloid (i.e., autologous monocytes fromPB) and lymphoid (EBV-transformed B cells) targets are not rec-ognized by our T cell effectors, suggesting the existence of leuke-mia-associated or leukemia-specific Ags amenable to identifica-tion. In two cases, we also could provide proof of principle that ourCTL were really effective in the killing of naive AML cells, sug-gesting that Ag recognition is not accompanied by any impairmentof CTL functional properties due to tumor counterattack, such asCTL apoptosis induced by FasL-expressing AML cells.

Finally, to be used in vivo, leukemic m-DC need to be irradiatedto prevent proliferation of residual undifferentiated leukemic cells.Therefore, leukemic DC should maintain their functional proper-ties also after irradiation. Notably, leukemia-specific autologous Tcells were obtained using irradiated leukemic DC as stimulatorsand this observation already provides evidence for their capacity toactivate T cells in vitro. In addition, leukemic i-DC that were ir-radiated 4 h after CD40L stimulation could still differentiate intofunctional competent m-DC, as shown by their capacity to up-modulate maturation markers such as CD83 and CCR7, to producecytokines/chemokines and to migrate in vitro up to 48 h after treat-

ment. For vaccine design purposes, though, it has to be kept inmind that a decrease in cell number occurs within the same timeframe. Dose, frequency, and route of DC delivery are crucial pa-rameters for vaccine effectiveness, which have not been optimizedyet in clinical trials with normal nonirradiated DC (37). Neverthe-less, based on our data, it is reasonable to anticipate that a highercell number should be used when vaccinating with leukemic m-DCas compared with normal DC, also because cells with m-DC phe-notype do not represent the totality of cells in bulk cultures ofleukemic DC. Tumor cell number is not a limitation in AML pa-tients, though, considering the relative ease of tumor cell acquisi-tion at disease presentation. The availability of large numbers ofleukemic cells also overcomes the issue of the low yield of viableleukemic m-DC at the end of the differentiating culture (6, 7, 38).In our series, cells with m-DC phenotype represented 10–50% ofthe initial number of input cells, which would enable (considering500 � 106 as the minimum number of leukemic cells obtainable atdiagnosis), the manufacturing of at least 10 vaccine preparations of5 � 106 cells/vaccine. The real limiting factor remains only thefact that differentiation into leukemic m-DC could be achievedonly in half of the patients analyzed. On one hand, a possiblesolution would be the prediction of which patients will generateleukemic DC and which patients will not. Recently, it has beenshown that the expression of CD14 (30) and of CD86 (20) onAML blasts correlates with their capacity of generating cells withDC features. In agreement with these observations, our data con-firm the predictive value of both markers and particularly of CD86,which was expressed by AML blasts in all 10 cases that coulddifferentiate to m-DC. This marker (alone or in combination withCD14) could be very helpful for determining which patients areeligible for vaccination. On the other hand, the addition of othercytokines to the GM-CSF/TNF-�/IL-4 mixture (for instance, stemcell factor and Flt-3) (13, 21–23) or the use of other compounds(such as phorbol esters or bryostatin) (26, 39) might increase thenumber of responsive cases.

To our knowledge this is the first report that studies the differ-entiating potential of leukemic i-DC into m-DC and provides fullfunctional characterization of leukemic m-DC from a preclinicalpoint of view. In addition, we show that leukemic m-DC efficientlyprime T cells from AML patients, generating MHC-restrictedCD4� and CD8� cells that are leukemia-specific. Our data suggestthat irradiated leukemic m-DC should be tested as a vaccine aimedat eradicating minimal residual disease in a significant fraction ofAML patients.

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