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Tumor Biology and Immunology

Development, Function, and Clinical Significanceof Plasmacytoid Dendritic Cells in ChronicMyeloid LeukemiaSabrina Inselmann1, Ying Wang1, Susanne Saussele2, Lea Fritz1, Christin Sch€utz1,Magdalena Huber3, Simone Liebler1, Thomas Ernst4, Dali Cai5, Sarah Botschek1,Cornelia Brendel1, Raffaele A. Calogero6, Dinko Pavlinic7, Vladimir Benes7, Edison T. Liu8,Andreas Neubauer1, Andreas Hochhaus4, and Andreas Burchert1

Abstract

Plasmacytoid dendritic cells (pDC) are the main producersof a key T-cell–stimulatory cytokine, IFNa, and critical regu-latorsof antiviral immunity.Chronicmyeloid leukemia(CML)is caused by BCR-ABL, which is an oncogenic tyrosine kinasethat can be effectively inhibited with ABL-selective tyrosinekinase inhibitors (TKI). BCR-ABL–induced suppression of thetranscription factor interferon regulatory factor 8 was previ-ously proposed to block pDC development and compromiseimmune surveillance inCML.Here, wedemonstrate that pDCsin newly diagnosed CML (CML-pDC) develop quantitativelynormal and are frequently positive for the costimulatory anti-gen CD86. They originate from low-level BCR-ABL–expressingprecursors. CML-pDCs also retain their competence to matu-rate and to secrete IFN. RNA sequencing reveals a stronginflammatory gene expression signature in CML-pDCs.

Patients with high CML-pDC counts at diagnosis achieveinferior rates of deep molecular remission (MR) under niloti-nib, unless nilotinib therapy is combined with IFN, whichstrongly suppresses circulating pDC counts. Although mostpDCsareBCR-ABL–negative inMR,a substantial proportionofBCR-ABLþCML-pDCspersists under TKI treatment. This couldbe of relevance, because CML-pDCs elicit CD8þ T cells, whichprotect wild-type mice from CML. Together, pDCs are identi-fied as novel functional DC population in CML, regulatingantileukemic immunity and treatment outcome in CML.

Significance:CML-pDC originates from low-level BCR-ABLexpressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatmentoutcome in CML. Cancer Res; 78(21); 6223–34. �2018 AACR.

IntroductionBCR-ABL1 is a fusion oncogene, which arises from the t(9;22)

(q34;q11) chromosomal translocation in a hematopoietic stemcell (1, 2). Chronicmyeloid leukemia (CML) seems to be sensitiveto immune therapy, such as with IFNa, which stimulates CML-specific T-cell responses (3, 4). Mounting of a specific T-cellresponse requires, first, recognition through the T-cell receptorof peptide–MHC class I complexes on the surface of antigen-presenting dendritic cells (DC). However, there is conflicting

evidence regarding the capability of BCR-ABL–expressing leuke-mic CML stem cell–derived DCs (5–9) to trigger CML-specificT-cell immunity.

Little is known about the origin, regulation, and function of aspecialized typeofDCs,plasmacytoidDCs(pDC) inCML.NormalpDCs are the main source of a key Th1 immune-stimulatorycytokine,IFN(10–12).pDCsregulateinnateandadaptiveimmuneresponses (12, 13) and contribute to immune activation (13), butalso tolerance(14,15).UntreatedpatientswithCMLwere reportedto have reduced pDC counts (16–18), which might involve BCR-ABL–mediated suppressionof interferon regulatory factor 8 (IRF8,ICSBP) gene expression (19–23), a supposed pDC-fate–definingtranscription factor (24, 25). Although BCR-ABL–induced Irf8suppression was linked to impaired pDC development based ona murine model of CML (20), recent genetic in vivo evidencedemonstrated that pDC development is Irf8-independent (26),essentiallyrequiringonlyE2-2(Tcf-4)andZeb-2(27,28).However,Irf8 is clearlyofkey importance for the functionofpDCs—themostprominent being IFNproduction (24–26). In a translational studyof themulticenter tyrosine kinase inhibitor (TKI) discontinuationtrial, EUROSKI, we recently observed unusual high counts ofmature (CD86þ) pDC in patients with CML in deep molecularremission(MR). Their abundancewasassociatedwithCD8þT-cellexhaustion and predictive of a significantly lower chance of sus-taining treatment-free remission (TFR) after TKI stop (29).

Here, we characterized the origin, genetic, and biological char-acteristics of pDCs in untreated CML (CML-pDCs) and corrob-orated specific aspects of CML-pDC development and function

1Department of Hematology, Oncology and Immunology, University HospitalGiessen and Marburg, Campus Marburg, Philipps University Marburg, Marburg,Germany. 2Department of Hematology/Oncology, University Hospital Man-nheim, University Heidelberg, Mannheim, Germany. 3Institute for Medical Micro-biology and Hospital Hygiene, University of Marburg, Marburg, Germany. 4Klinikf€ur Innere Medizin II, H€amatologie und Internistische Onkologie, Jena, Germany.5Department of Hematology, First Affiliated Hospital, China Medical University,Shenyang, China. 6University Turin, Bioinformatics and Genomics Unit, Turin,Italy. 7Genomics Core Facility, European Molecular Biology Laboratory (EMBL),Heidelberg, Germany. 8The Jackson Laboratory, Bar Harbor, Maine.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Andreas Burchert, Universit€atsklinikum Giessen andMarburg, Onkologie und Immunologie, Marburg 35043, Germany. Phone:49-6421-5865611; Fax: 49-6421-5865613; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-1477

�2018 American Association for Cancer Research.

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using a CMLmouse model. Furthermore, we prospectively inves-tigated qualitative and quantitative pDC changes under nilotinibversus nilotinib plus IFN therapy within a large multicenter CMLtrial. We find that CML-pDCs emerge from low-level BCR-ABLþ

stem cells, produce inflammatory cytokines, and retain importantfunctional properties, such as IFN secretion, maturation, andelicitation of CML-specific immunity. Data support for the firsttime that CML-pDCs are a source of inflammation in CML andlikely involved in the regulation of treatment response andcontrol CML-specific immunity.

Materials and MethodsMice and cell lines

WT C57BL/6J mice, male at 6 to 10 weeks of age and femalemice at 12 to 14weeks of age,were used. Animal experimentswereperformed according toGerman lawandapprovedby the regionalboard "Regierungspr€asidium Giessen" (animal proposal # V54-19c20-15(1)MR20-36 Nr.07/2010 and # V54-19c2015h01MRNr47/2014). 32D cell line was purchased from the "DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH"(DSMZ), and Ph€onix eco cell line was obtained from AlleleBiotechnology. Cells were tested periodically and confirmed asMycoplasma free by the PCR-based method (myco: 50-gggacgaaa-caggattagataccct-30 and 50-tgcaccatctgtcactccgttaacctc-30), andauthentication was not conducted, unless by morphology checkdue to microscope. Both cell lines were maintained according tothe supplier's recommendations and not cultured more than2 months.

PlasmidsThe retroviral vectors pMIGp210 (encoding the BCR-ABLp210

and gfp), referred to as BCR-ABL, and pMIG (control, encodingonly gfp) were a kind gift from Dr. W. Pear (The Universityof Pennsylvania, Philadelphia, PA; ref. 30). Using this vector asbasis, the [BCR-ABL]-2A-[IRF8]-2A-[GFP] plasmid (BARF) wasconstructed, coexpressing BCR-ABL and Irf8 and gfp. Retroviralparticles were generated with Phoenix eco cell line as describedpreviously (31).

Generation of pDCs, immunization, and adoptive T-celltransfer

Bone marrow of wild-type mice was harvested, enriched forpDCs(pDCisolationkit II,Miltenyi), andspinoculatedwitheitherBCR-ABL– or BARF-containing retroviral particles. During spino-culation, pDCswere cultured in RPMIMedium1640 (Gibco) andsupplemented with 10% FCS and 1% penicillin/streptomycin, 20mmol/L HEPES, 1 mmol/L sodiumpyruvat, 50 mmol/L mercap-toethanol (all Gibco), and 100 ng/mL FLT3-ligand (FLT3-L;Roche). GFP-positive pDCs were sorted and then i.v. injected intothe tail vein of wild-type recipient mice (1,000–3,000 cells). pDCinjections were repeated weekly in each mouse, for 3 times.

CD8þ T cells were enriched at a purity of up to 80% fromspleens of pDC-immunized animals, and 2.5� 106 of these wereadoptively transferred i.p. the day after CML stem cell transplan-tation of independent recipient animals. To control for engraft-ment, blood samples frommice tail vainwere analyzedweekly forproportions of GFPþ cells.

Western blottingWestern blotting of 32D cells and primary murine cells was

performed as previously described. The following primary anti-

bodies were used: anti-IRF8 goat, clone C-19 and anti-c-Ablmouse, clone 24-11 (both Santa Cruz Biotechnology) and anti-Actin mouse, clone AC-15 (Sigma-Aldrich). The secondary anti-bodies were horseradish peroxidase–conjugated polyclonal rab-bit anti-goat and polyclonal goat anti-mouse antibodies (bothDako).

Primary CML blood samplesPeripheral blood samples of patients with primary diagnosis of

CMLor during follow-upwere received through the immunologicsubstudy of the German multicenter CML-V study. In accordanceto the Declaration of Helsinki, all patients gave written informedconsent to participate in this substudy of CML-V or EUROSKI.We confirm that both studies were approved by the localEthic Committees [NCT01657604 (CML-V) and NCT01596114(EUROSKI)]. Primary blood mononuclear cells (PBMC) wereisolated by Biocoll density gradient separation (density1.077 g/mL, Biochrom).

Flow cytometry analysis and cell sortingFlow cytometry was performed on the LSRII (BDBiosciences),

and data were analyzed using the FlowJo software (FlowJo LLC).For fluorescence-activated cell sorts, cell populations wereenriched prior to sorting using magnetic bead enrichment (Mil-tenyi). Sorting was performed using the MoFlo XDP (BeckmanCoulter). For analysis of maturation status, the PBMCs werecultured for at least 18 hours with and without 2 mmol/L CpG2006 in RPMI Medium 1640 (Gibco) and supplemented with10% FCS and 1% penicillin/streptomycin, prior to antibodystaining. Two four-color stainings were performed for each,untreated and treated cells, which included FcR blocking,BDCA-2 (PE), CD123 (FITC), and eitherHLA-DR (APC)þCD86(TRI-COLOR) or CD80 (APC) þ CD40 (TRI-COLOR). Due tovery low cell numbers, isotype controls for APC- and TRI-COLOR stainings were performed onCpG-treated cells and usedfor both untreated and treated cultures. A list of used antibodiesand enrichment kits is attached (Supplementary Tables S1 andS2). For details on flow cytometry (sample preparation,gating, and abundance of populations), see SupplementaryTable S3.

Intracellular IFN stainingFor intracellular staining of primary human pDCs, enriched

cells (Supplementary Table S2) were cultured in 100 mL RPMIMedium 1640 (Gibco) and supplemented with 10% FCS and 1%penicillin/streptomycin, in 96-well U-bottom plates for 5 hourswith and without 5 mmol/L CpG 2216 (TIB MOLBIOL). After2-hour incubation, 1 mL Golgi-Plug (BD Biosciences) was addedinto each well. Then the cells were stained for surfacemarkers andafterward fixed with 100 mL Medium A for 15 minutes at roomtemperature (dark). This was followed by permeabilization with100 mL Medium B (both Thermo Fisher Scientific) and simulta-neous staining with the intracellular IFNa antibody, FITC-conjugated (clone 225.C, Chromaprobe) for 20 minutes at 4�C.The cells were resuspended in 2% formaldehyde (Fischer) andanalyzed on the LSRII.

CML transduction/transplantation modelBone marrow of wild-type mice was harvested by flushing

femura and tibiae. Mononuclear cells were obtained after densi-ty-gradient centrifugation (density 1.086 g/mL, Pancoll, PAN-Biotech). Stem cells for transplantation were enriched from bone

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marrow mononuclear cells using immunomagnetic column sep-aration (lineage depletion) of lineage-positive cells (Lineage CellDepletion Kit,Miltenyi) and then enrichment of Sca-1þ cells (easysep mouse SCA1-positive selection kit, stem cell). Isolated stemcells had a purity of 85% to 95%, were precultured for 18 hours inIMDM (Gibco, Thermo Fisher Scientific), and supplementedwith 20% FCS, 1% penicillin/streptomycin (both PAN Biotech),50 mmol/L Mercaptoethanol (Gibco), 10 ng/mL recombinantmurine IL3, 50 ng/mL recombinant IL6, and 50 ng/mL recom-binant murine SCF (all Immunotools). Cells were transducedwith either pMIG, BCR-ABL-, or BARF-retroviral particles byspinoculation (in the presence of 5 mg/mL polybrene) for 2consecutive days. GFP positivity was assessed by flow cytometry.Note that 3 � 104 GFPþ cells were injected intravenously via tailvein into sublethally (7 Gy) irradiated female recipient mice.Animal experiments were performed according to Germanlaw and approved by the regional board "Regierungspr€asidiumGiessen" (animal proposal # V54-19c20-15(1)MR20-36 Nr.07/2010 and # V54-19c2015h01MR Nr47/2014). For details regard-ing animal experimentation (sample size calculation, data exclu-sion, replication, randomization, and blinding), see Supplemen-tary Table S4.

BCR-ABL FISHBCR-ABL FISH was performed as previously described (32).

Quantitative real-time PCRMurine RNA was isolated and reverse transcribed using

RNeasy Micro Kit and Omniscript RT Kit (both Qiagen) accord-ing to the manufacturer's protocol. RNA of human samples wasisolated and transcribed using TriFast (peqlab) and SuperScriptIII reverse transcriptase (Thermo Fisher Scientific). Real-timequantitative PCR data for IRF8 gene expression were performedusing the DDCt method, with GAPDH as the housekeeping gene.Samples were analyzed in triplicates using QuantiTect SYBRGreen Mix (Qiagen). The following primer pairs were used:mouse gapdh: 50-catggccttccgtgttccta-30 and 50-cctgcttcac-caccttcttgat-30; mouse irf8: 50-tgccggcaagcaggattaca-30 and 50-ccacgtggctggttcagctt-30; human gapdh: 50-ctcctccacctttgacgctg-30 and 50-accaccctgttgctgtagcc-30; human irf8: 50-gtcccaactgga-catttccg-30 and 50-cattcacgcagccagcag-30. Quantitation ofBCR-ABL levels in patients was performed following proceduresand definitions of molecular response as previously described.Results were reported analogous to the European LeukemiaNetcriteria as a normalized transcript number of the internationalscale, IS (33). Absolute BCR-ABL transcript numbers wereassessed relative to GUS expression.

For competitive BCR-ABL and gfp PCR, we used genomic DNAof either spleen or chloroma tissue. For amplification, we usedDreamTaq DNA Polymerase (Thermo Fisher Scientific), andthe following 2 primer pairs: ba-1 50-ttcagaagcttctccctggcatccgt-30 and a-1 50-ggtaccaggagtgtttctccagactg-30, generating a 487 bpfragment, and gfp-for 50-cgtaaacggccacaagttca-30 and gfp-rev 50-tcttgtagttgccgtcgtcc-30, generating a 260 bp product.

CytologyCytological smears were stained by the panoptic method of

Pappenheim and analyzed using the Olympus BH-2 microscope(Olympus), oil objective,�100. Images were done with the DHSpicture database software (Dietermann & Heuser SolutionGmbH).

PCR amplification and sequencing of mutationsNext-generation sequencing of ASXL1 from genomic DNA of

CD34þ enriched cells was performed as described previously(34). For conventional sequencing, gDNA from sorted pDCs andCD34þ/CD38þ cells was preamplified with GoTaq DNA Poly-merase (Promega) using the following primers: human ASXL1mut2: 50-cacttacaaaagaccagagcca-30 and 50-ctggatggagggagtcaaaa-30. The resulting 237 bp DNA product was cleaned up by theNucleoSpin Gel and PCR Clean-up Kit (Machery and Nagel) andthen sequenced with the human ASXL1 mut2 reverse primer bySeqlab. In a patient harboring the BCR-ABL F359I mutation,cDNA of FACS-sorted pDCs and PBMCs was used in PCR withPhusion Green High Fidelity DNA-Polymerase (Thermo FisherScientific). The primers of ABL-A, 50-acagcattccgctgaccatcaataag-30

and 50-atggtccagaggatcgctctct-30, amplified a 1,718 bp fragment.Using this fragment, a nested PCRwasperformedwith the primerspair: human BCR-ABL F359I 50-catgacctacgggaacctcc-30 and50-ggccaaaatcagctaccttc-30. A 200 bp PCR product was sequencedwith the BCR-ABL F359I forward primer.

Sequences were analyzed for mutations using the BLAST Alignsoftware (www.ncbi.nlm.nih.gov/BLAST/) and are shown aselectropherogram.

mRNA sequencingTotal RNAs were isolated from sorted pDCs of healthy donors

and the first diagnostic patients with CML with RNeasy Micro Kit(Qiagen), according to themanufacturer's protocol. RNA librarieswere generated with the NEBNext Ultra RNA Library Prep Kit forIllumina (New England Biolabs Inc.), according to the manufac-turer's protocol. The libraries were sequenced on an IlluminaHiSeq 2000, requested Read Length HiSeq 2� 75 bp with pairedend.

Statistical methodsData were analyzed with GraphPad Prism 6 software

(GraphPad Software, Inc.). Statistical significance of data wasanalyzed by the unpaired t test or ANOVA, after testing for normaldistribution, unless indicated otherwise. Significance of survivaldifference was analyzed by the Mantel–Cox test. P value isshown as: ns, nonsignificant, P > 0.05; �, P < 0.05; ��, P < 0.01;��, P < 0.001; and ��, P < 0.0001.

SoftwareFlow cytometry data were acquired with BD FACSDIVA soft-

ware (Version 6.1.3; BD Biosciences) and analyzed by FlowJosoftware (either onwindows version 7.6 orMac version 9.5) fromFlowJo LLC.

The Summit software (version 4.1; Cytomation) was used foracquiring cell sorts.

For Pappenheim image acquisition and processing, the DHSpicture database software was used (Dietermann & Heuser Solu-tion GmbH).

Statistical analysis was performed with GraphPad Prism 6software (GraphPad Software, Inc.).

ResultsCML-pDCs are BCR-ABL–positive, express IRF8, and develop atnormal frequency

Patients with untreated CML were previously reported to havestrongly reduced pDC counts (16–18). However, here we show

Inflammatory BCR-ABLþ Plasmacytoid Dendritic Cells

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www.ncbi.nlm.nih.gov/BLAST/


that patients with treatment-na€�ve CML only have lower pDCproportions in the peripheral blood (% pDC in white bloodcells; Fig. 1A and B), whereas their absolute pDC numbers (pDCsper mL) are not reduced (Fig. 1C). Moreover, we found that CML-pDCs are BCR-ABL–positive (BCR-ABLþ) by FISH (median: 81%;range: 57%–100%, n¼ 8; Fig. 1D). They also express normal IRF8levels when compared with normal donor pDCs (Fig. 1E). Thus,human data do not show a link between BCR-ABL, IRF8 expres-sion, and pDC counts in CML.

CML-pDCs develop from low-level BCR-ABL–expressingstem cells

To study the origin of CML-pDCs and a previously proposeddependence of CML-pDC development on Irf8, we askedwhetherablockofCML-pDC transdifferentiation from leukemic stemcellsis caused by BCR-ABL–mediated Irf8 suppression in vivo. Themurine transduction/transplantation (tt)-CML model wasemployed (35). In the tt-CML mouse model, leukemogenesis

depends on the engraftment of BCR-ABL–transformedLin�Sca1þc-Kitþ cells (LSK; refs. 36, 37). BCR-ABLþ hematopoi-esis can be tracked using GFP expression as a marker. Irf8 wasstrongly reduced by BCR-ABLþ in CML stem cells (CML-LSK),common lymphoid (c-KitþSca-1þIL7-receptor alphaþGFPþ;CLP), and myeloid progenitors (c-KitþSca-1�CD16/32�CD34þGFPþ; CMP; Supplementary Fig. S1A), but, in contrastto patients with CML (Fig. 1C), CML mice showed a severe pDCloss (Supplementary Fig. S1B). Moreover, restoring IRF8 expres-sion in BCR-ABLþ CML stem cells in vivo using a BCR-ABL/Irf8coexpression construct (BARF; Fig. 2A) did not rescue CML-pDCmaturation, suggesting that the CML-pDC maturation block iscausally linked to BCR-ABL overexpression, but not IRF8 sup-pression (Fig. 2B). This was further supported by the finding thatBCR-ABL caused an expansion of CML-LSK, T cells (CD3þ), B cells(B220þ), and myeloid cells (Gr1þ), but selectively blocked mat-uration of pDCs irrespective of restored Irf8 levels (Fig. 2C). Ofnote, the few GFPþ pDCs, which transdifferentiated from GFPþ

Figure 1.

CML-pDC: frequency, BCR-ABL–positive origin, and IRF8 expression. A, Representative staining of PBMCs for BDCA-2þ/CD123þ pDCs in a patient with untreatedchronic phase CML versus a normal donor.B, Percentage of pDCs in peripheral blood of normal donors versus patientswith untreated CMLwithin the CML-V study.C,Absolute pDCs counts (pDCs/mL) of cohorts as in B.D, BCR-ABL interphase FISH of patients with untreated CML. Top, representative FISH staining of pB CML-PBMCand CML-pDCs (yellow, BCR-ABL fusion signal); bottom, quantitation of BCR-ABL FISH-positive cells and pDCs frequency in peripheral blood (pB; analyzedcell nuclei: 20 to up to 154). E, IRF8mRNA expression of sorted human BDCA-2þ/CD123þ pDCs from normal donor (n¼ 3) and patients with untreated CML (n¼ 10).Asterisks indicate significance level, �� , P < 0.0001 or ns, nonsignificant, P > 0.05 according to Mann–Whitney test (B and C) or unpaired t test (E).

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Figure 2.

pDCs show low-level BCR-ABL expression. A, Left, retroviral BARF-coexpression construct; middle, Western blotting of BARF-transduced 32D cl cell line; right,Western blotting of protein lysates from CMLBCR-ABL and CMLBARF mice (spleen cells). B, Left, absolute number of mPDCA-1þ/B220þ pDCs developing from GFPþ

hematopoiesis in transplanted mice for spleen; right, bone marrow. Note that one proportion of CMLBARF mice succumb on a myeloproliferative CML-like disease,referred to as CML-BARF(lethal), and another proportion survives (CML-BARF(surviving)). C, Extent of BCR-ABL–positive (GFPþ) hematopoiesis in bone marrow in theprimitive LSC fraction and distribution in various terminally differentiated compartments for CMLBARF (lethal), CMLBCR-ABL, and control mice. The indicatedcompartments were gated as Lin�Sca1þc-Kitþ (LSK), GR-1þ, CD3þ, B220þ, and mPDCA1þ/B220þ (pDCs) and then analyzed for percentage of GFPþ cells. D, FACShistograms illustrating mean fluorescence intensity (MFI) of GFP (BCR-ABL) of BCR-ABLþ pDCs versus BCR-ABLþ total cells of six CML mice (n ¼ 15). E, Foldreduction of absolute BCR-ABL transcript numbers measured by real-time PCR and expressed as a BCR-ABL/GUS ratio in sorted CML-pDCs compared with thedenominator total white blood cells of the same patient, respectively. F, Left, FACS analysis BDCA-2þ/CD123þ pDC frequency and BCR-ABLIS in pB of CML patient#059-505 at different time points of nilotinib treatment; right, Sanger sequencing electropherogram of cDNA from sorted pDCs and PBMC of a de novo emergingF359I nilotinib resistancemutation in BCR-ABL.G, Left, FACS-sorting strategy of patient #013-501; right, Sanger sequencing electropherogram of cDNA from sortedCD34 cells for an ASXL1 frame shift mutation and the absence of it in sorted pDCs fraction. Asterisks indicate significance level, � , P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001; or ns, nonsignificant, P > 0.05 according to one-way ANOVA with Tukey multiple comparison test (B) or two-way ANOVA with Tukey multiplecomparison test (C), unpaired t test (D), or paired t test (E).






stem cells, consistently expressed much lower BCR-ABL levels(GFP) than the remaining leukemic bulk in the same animals (Fig.2D). Likewise, human CML-pDCs expressed in median 3.5-foldlower BCR-ABL mRNA (range: 1.2–6.8) than the leukemic bulk(Fig. 2E). This supports that high BCR-ABL expression thresholds,not IRF8 loss, suppress CML-pDCmaturation.We concluded thatthe reasonofmaintainedCML-pDCmaturation inhumanCMLasopposed toCMLmice is clonalBCR-ABL expression heterogeneity(31, 38, 39). CML-pDCs likely originate from stem cells withlower-level BCR-ABL expression, which is rare in tt-CML mice.Indeed, a heterogeneous evolutional trajectory of CML bulkcells and CML-pDCs was supported genetically in 2 patients withCML. One patient carried an ABL-resistance mutation, detectableonly in the sorted CML bulk, but not in the sorted pDC fraction(Fig. 2F). The other patient had an ASXL1 mutation that wasdetectable in sorted CML-CD34þ cells, but not in CML-pDCs(Fig. 2G).

CML-pDCs display constitutive inflammatory signalingIn order to obtain insights into the activation- and functional

status of CML-pDCs, we compared the gene expression profiles ofCML-pDCs and normal donor pDCs using RNA sequencing(Fig. 3A). Gene expression profiles of CML-pDCs (n¼ 6) differedsignificantly from normal pDCs (n ¼ 3; Fig. 3B; SupplementaryTable S5). With a median sequencing depth of 127.2 � 106

mapped reads (CML-pDC samples) and 154.1 � 106 mappedreads in normal pDC samples, 3,109 genes were found to besignificantly regulated (log2FC � 1) in CML-pDCs (Fig. 3B;Supplementary Data File S1). The majority of these differentiallyregulated genes (63.5%) were upregulated (Fig. 3C). Significantlymore of these genes (20.5%) were strongly upregulated in CML-pDCs, whereas only a minority (5.8%) were strongly downregu-lated. Gene set enrichment analysis (40) showed enrichment inCML-pDCs of nine classes of genes involved in antigen capturing,processing, and presentation byMHC class I and II, as well as IFN,

Figure 3.

Inflammatory signaling in CML-pDC. A, Sort strategy and Pappenheim staining of sorted human normal and untreated CML BDCA-2þ/CD123þ pDCs formRNA sequencing. B, Hierarchical clustering of the set of genes detected as significantly differentially expressed between CML-pDCs and normal donor pDCs.Hierarchical clustering parameters: Euclidean distance, average linkage. C, Pie chart illustrating extent of differential expression between CML-pDCsand normal donor pDCs. D, Top, leading edge analysis of the nine immune-related GSEA classes, which are highlighted in bold (bottom), reveals subsets ofgenes that contribute most to the enrichment result. Bottom, all significantly enriched immune-related GSEA classes in CML-pDCs versus normal donor pDCs.

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IL, and Toll-like receptor (TLR) signaling (Fig. 3D). The leading-edge subsets of genes in these nine classes were commonly over-lapping between 2, 3, 4, or more classes, consistent with theircritical importance (Fig. 3D; Supplementary Data File S2).

CML-pDCs are functional, frequently CD86þ, and overexpressinflammatory cytokines

IFN secretion is a key function of activated normal pDCs andIrf8-dependent (26, 27). mRNA sequencing shows thatCML-pDCs constitutively express higher basal levels of IFNsthan normal donor pDCs in an IRF8-dependent manner(Fig. 4A). IFN secretion is also promptly induced upon in vitrostimulation with the TLR9 ligand (oligonucleotide CpG2216; Fig. 4B). In contrast, healthy donor-derived pDCs do notexpress or secrete IFN unless stimulated with CpG (Fig. 4A andB). In addition, also other cytokines, which are implemented inthe elicitation of T-cell responses (e.g. ILs, CXCL9, 10, 11, CCL

22) or CML stem cell survival (TNFa, TGFb; refs. 37, 39, 41), areoverexpressed in CML-pDCs. TGFb is strongly expressed also innormal pDCs (Fig. 4C). Intriguingly, although normal pDCsmature and then upregulate MHC class II and T-cell costimula-tory molecules (CD80, CD86, and CD40) in response to CpG-induced TLR signaling (Fig. 4D, top), CML-pDCs are frequentlyspontaneously mature/activated (CD86þ, CD40þ) and thusresemble a recently described subpopulation of normalBDCA-2þCD123þ pDCs, so-called AxlþSiglec-6þDCs (AS-DCs)or pre-DCs (Figs. 3A and 4D, bottom; Supplementary Fig. S2;refs. 42, 43) and display activated TNF, JAK, andNF-kB signaling(Supplementary Fig. S3), suggesting a constitutive autocrinestimulation of CML-pDCs by secretion of TNF, IFNs, and ILs.Only aminority of patients with de novoCML (exemplary shownpat. # 009-501) do not show spontaneous maturation andCD86, CD80, or CD40 expression (Supplementary Fig. S2).However, also immature CML-pDCs (pat. #009-501) retain

Figure 4.

CML-pDCs are functional and express high amounts of inflammatory cytokines. A, Heat map, based on RNA-seq data [transcripts per million (TPM)],illustrating the expression of IFNa subtypes in untreated CML-pDC and normal donor pDCs. Samples are ordered for IRF8 expression, and individualsample ID is mentioned at the bottom. B, Left and middle, representative FACS plots showing intracellular IFNa staining of MACS-enriched pDCs with and withoutTLR-9 engagement by in vitro CpG treatment. Exemplary shown are two normal donors (left) and patients with untreated CML (middle); right, statistical analysis ofproportion of IFN-secreting pDCs.C,Heatmaps, basedonRNA-seqdata (TPM), illustrating expressionof different cytokines in untreatedCML-pDCand normal donorpDCs. Individual sample ID is mentioned at the bottom. D, FACS histogram of activation/maturation markers of pDC (gated as CD123þ/BDCA-2þ) with and withoutCpG treatment for a normal donor (top plot) and a CML-patient # 001-509 (bottom plot). Shown are the indicated specific antibodies (gray curves) in comparisonwith isotype controls (black curves). Same isotype controls were used for untreated andCpG-treated cultures. Asterisks indicate significance level, � , P <0.05 or �� , P< 0.01 according to two-tailed unpaired t test (B).






their IFN secretion capacity and mature upon CpG treatment(Supplementary Fig. S2).

ABL-TKI blocks CML-pDC development and suppresses pDCnumbers in remission

In vivo treatment with the BCR-ABL–specific TKI nilotinibreduced CML-pDC counts within months of therapy (Fig. 5A).This suggests an antiproliferative effect of TKI on CML-pDCprecursors. A more potent reduction in pDC counts occurredunder cotreatment with nilotinib and IFN within the CML-V trial(Fig. 5A). This combined therapy also reduced the number ofmature (CD86þ) pDCmore efficacious than nilotinib alone. Thiscould be clinically important, because CD86þpDCs were previ-ously linked to a lower TFR probability (29). Of note, there is arapiddecline of theCD86þpDCcounts after 6 to12months under

nilotinib and more pronounced with nilotinib plus IFN.CD86þpDC counts were comparably low in patients withlong-term imatinib treatment (median, 7.5 years) within theEUROSKI TKI discontinuation trial (Fig. 5A and B). More than10 CML-pDCs/mL at diagnosis—corresponding to the mediannumber of pDC detected inMR (Fig. 5A)—were associated with areduced probability to achieve a MR4 after 12 months of therapywith nilotinib (Fig. 5C). Interestingly, if patients with CML-pDCcounts > 10/mL were randomized to receive nilotinib plus IFN, aninferior molecular response was no longer seen (Fig. 5C). PDCsfrom patients in MR were BCR-ABL–negative by FISH (n ¼ 8/8),which enabled to analyze approximately only 100 pDCs perpatient (Fig. 5D). However, when using absolute quantificationand a standardized, international scale BCR-ABLIS PCR (sensitiv-ity 5 � 10�5), a median of 7,700 BCR-ABL copies (range,

Figure 5.

Remission pDC counts are suppressed and impaired CML-pDC development. A, Absolute pDC counts (pDCs/mL) in peripheral blood of patients withuntreated CML compared with nilotinib or nilotinib þ PEG IFN treatment for 6 to 12 months within the CML-V study, or versus long-term imatinib-treated patientsfrom EUROSKI study. Dotted line indicates mean level of absolute pDC in normal donors. B, Absolute CD86þpDCs counts (pDCs/mL) of cohorts as in A. Dotted lineindicates mean level of absolute CD86þpDC in normal donors. C, Analysis for achieving a BCR-ABLIS of � 0.01 % after 12 months of therapy according totheir absolute pDC counts (pDC/mL) and their therapy-arm within CML-V study. White numbers, patients who achieved BCR-ABLIS of � 0.01%; blacknumbers, total patients. D, BCR-ABL interphase FISH of remission pDCs from deep MR of CML. Top, shown are representative cell pictures; bottom, quantitation ofBCR-ABL FISH-positive cells and pDCs' frequency in peripheral blood (pB; analyzed cell number: 100, except R56, 89 cells). E, BCR-ABL (BCR-ABL/ABL ratio)expression levels in pDCs of remission patient in comparisonwith peripheral blood from the same sample are shown. Asterisks indicate significance level, �, P <0.05;�� , P < 0.01; ��, P < 0.0001; or ns, nonsignificant, P > 0.05 according to Kruskal–Wallis test with Dunn multiple comparison (A and B) or Fisher exact test (C) orunpaired t test (E).

Inselmann et al.





470–17,000) were detected in every patient. Thus, numbers ofresidual CML-pDCs remain detectable by PCR in all 18 analyzedpatients—even in very deep MR of CML (Fig. 5E). This suggeststhat a significant proportion of BCR-ABLþ CML-pDCs isTKI-insensitive.

BCR-ABL does not inhibit the in vivo T-cell priming functionof pDCs

To experimentally assess the functional competence and bio-logical relevance of persisting BCR-ABLþ pDCs in vivo, wild-typeC57BL/6Jmice were vaccinated with BCR-ABLþ pDCs or differenttypes of control pDCs. Vaccination occurred intravenouslyweeklyfor 3 successiveweeks using 1 to 3� 103 in vitro–generated controlpDCs (pDCwt), BCR-ABLþ pDCs (pDCBCR-ABL), BARFþ pDCs

(pDCBARF), or gfpþ Mig-control pDCs (pDCMig), respectively(Fig. 6A).

CD8þ cytotoxic T cells from these vaccinated mice (TcBCR-ABL,TcBARF, Tcwt, and TcMig) were sorted and adoptively transferredinto other recipients: tt-CML-mice (Fig. 6A). These mice suc-cumbed from GFPþ leukemia, unless they had received TcBCR-ABL

or TcBARF

, which potently suppressed the outgrowth GFPþ leuke-mia (Fig. 6B–D).Ofnote, pDCMig,whichdonot expressBCR-ABL,but only gfp, also primed a leukemia-protective T-cell response.Presumably, GFP is the target of this T-cell response, becauseleukemic relapse after TcMig transfer was gfp-negative and BCR-ABL–positive, suggesting an immune escape through loss of gfpexpression. In contrast, mice relapsing despite TcBCR-ABL or TcBARF

lymphocyte infusions were never gfp-negative (Supplementary

Figure 6.

CML-pDCs can induce protective T-cell immunity in CML. A, pDC immunization mouse model. pDC generation: in vitro generation of BCR-ABLþ pDCs byretroviral transduction of enriched pDCs with either BCR-ABL, BARF, or empty Mig-vector; pDC immunization: injection of sorted BARFþpDCs, BCR-ABLþ

pDCs, untransduced pDCs, or Mig-vector–transduced pDCs intoWTmice once weekly for three successive weeks; adoptive T-cell transfer: isolation and injection ofCD8þ cytotoxic T cells from immunized mice into BCR-ABL–transplanted mice on day þ1. B, Representative FACS plots of kinetics of GFP (BCR-ABL)–positivecell proportions in blood of CML mice after adoptive T-cell transfer from pDCBCR-ABL-, pDCBARF-, pDCwt-, or pDCMig-immunized mice at indicated time points.C, Summary of GFP-positivity kinetics in blood from surviving pDCBCR-ABL mice, surviving pDCBARF, moribund pDCwt mice, and surviving pDCMig over time.D, Survival curves of CML mice after adoptive T-cell transfer from pDCBCR-ABL-, pDCBARF-, pDCwt-, and pDCMig-immunized mice. P value was assessed by theMantel–Cox test.






Fig. S4). Thiswould be in linewith an immune escape, supportingthat BCR-ABLþ pDCs could be functional regulators of CMLimmunity. However, it cannot be ruled out that GFP-derivedneoantigens or BCR-ABL–dependent leukemia-associated anti-gens are also released from GFPþ dying cells and then cross-presented by the recipient's own professional DCs such as cDCs,but also pDCs.

DiscussionBCR-ABL–induced IRF8 suppression has previously been caus-

ally linked to pDC loss, pDC dysfunction, and a lack of immunesurveillance in CML (16–18, 20, 44). Our results do not supportthis concept. First, while acknowledging that BCR-ABL overex-pression suppresses Irf8 in primitive murine hematopoiesis andabrogates pDC development from BCR-ABLþ LSCs in mice (Fig.2B and C; Supplementary Fig. S1; refs. 20, 45), there is no causallink between IRF8 loss and pDC loss in vivo. Secondly, there is nopDC loss in human CML, although CML-pDCs are of BCR-ABL–positive origin (Figs. 1C and 5A).

Data rather support that modeling human CML-pDC devel-opment in tt-CML mice is flawed by BCR-ABL overexpression,which is a prerequisite for LSC transformation in mice andpotently abrogates pDC transdifferentiation from LSC(Fig. 2D). In contrast, CML-stem cell evolution is characterizedby clonal heterogeneity (38, 39, 46), including BCR-ABL expres-sion variability (31). Data imply, therefore, that pDC develop-ment in human CML is maintained from low-level BCR-ABL–expressing precursors, which are not progenies of the CML LSCclone producing the bulk of leukemia cells (Fig. 2F and G). Here,we showed that in spite of their BCR-ABLþ origin, CML-pDCsretain important functional pDC-properties, namely, IFN secre-tion and maturation capacity. At the same time, CML-pDCsaberrantly express multiple inflammatory cytokines and chemo-kines. Autocrine constitutive activation of JAK-STAT signaling inCML-pDCs could be a consequence (Supplementary Fig. S3),possibly leading to their peculiar mature phenotype in steadystate (Fig. 4D; Supplementary Fig. S2). Very recently, a subpop-ulation of BDCA-2þ CD123þAxlþSiglec-6þ pDCs (AS-DC) alsoreferred to as pre-DCs have been identified in normal individuals.These cells are very potent CD4þ and CD8þ T-cell stimulators.This raises the intriguing possibility that BCR-ABL leads toan expansion of BDCA-2þCD123þ CML-pDCs, which are

AS-DC-/pre–DC-like and hence, unlike BDCA-2þCD123þ con-ventional pDCs, T-cell stimulatory (42, 43).

Via paracrine effects, CML-pDC–derived TNF and tumor-derived growth factor (TGF) might also promote CML stem cellsurvival (47, 48), limit TKI response, and contribute to CML-stem cell persistence (39). Supporting this, we noticed that higherCML-pDC counts at diagnosis were associated with an inferiordepth of nilotinib response. However, when patients with highCML-pDCs at diagnosis were treated with a nilotinib/IFN com-bination within the randomized German CML-V study, totalpDCs including CD86þpDCs were strongly reduced, and anassociation between high CML-pDC counts at diagnosis andinferior depth of response was no longer seen. Based on ourrecent results showing that CD86þpDCs are associatedwith lowerprobability of TFR after TKI cessation (29), this implies that an IFNplus nilotinib-based induction therapy leads to a higher rate ofTFR, which is the primary endpoint of the CML-V study(NCT01657604). To study this, it will be tested whether TFR riskis associated with the number of CD86þpDCs and exhausted Tcells measured at diagnosis and prior to TKI stop.

Chronic inflammation has detrimental effects during tumori-genesis. It promotes tumor evolution and is immune suppressive.We showed here that low-level BCR-ABL–expressing CMLstem cells might trigger chronic inflammation by transdifferentia-tion into CML-pDCs as a source for many inflammatory chemo-kines and cytokines. Indeed, there is precedence that persistenthigh counts of activatedCD86þpDCs during chronic infection areimmune suppressive.Mechanistically, pDCs can induce immune-suppressive regulatory T cells (15, 49) and attenuate naturalkiller– and T-cell functions (50).

In contrast, in the early (preleukemic) evolution of CML, lowfrequencies of BCR-ABLþ pDCs might be tumor-suppressive.When low numbers of BCR-ABLþ pDC were injected into wild-type mice, they acted as vaccines and induced antileukemicT cells, which indeed protected from leukemia in vivo, whenadoptively transferred into CML mice. This suggests that CML-CD86þpDCs—like their normal AS-pDC counterparts—are capa-ble of priming T-cell responses to endogenously expressed anti-gens (12, 51, 52). Because a significant proportion of CML-pDCsare TKI-insensitive and persist during TKI-induced MR (Fig. 7),we propose that they elicit anti-CML CD8þ T-cell responses inMR via exploiting a restored and less exhausted remission T-cellrepertoire (53, 54). It will now be important to validate that

Figure 7.

Proposed roles of inflammatory CML-pDCs in immunityand treatment response of CML. Hematopoietic stemcells acquire the BCR-ABL translocation and initiallyexpress low-level BCR-ABL and thus retain the ability totransdifferentiate into CML-pDCs. These cells aberrantlyexpress inflammatory chemokines, cytokines, and CML-stem cell–protective TNFa and TGFb. Whereas limitednumbers of inflammatory pDCsmay trigger antileukemicimmunity during the early phase of CML evolution andagain in deep MR under TKI-based treatment, highCML-pDC counts (expanding with BCR-ABLþ stem cellmass) drive chronic inflammation, which stimulates CML-stem cell persistence and immune suppression.

Inselmann et al.





BDCA-2þCD123þCD86þ CML-pDCs correspond to theCD86þAS-DC/pre-DC population (42, 43). Beyond the scope ofCML, it is tempting to speculate that other oncogenic drivermutations, which lead to an increased stem cell fitness and stemcell expansion such as in benign clonal hematopoiesis (55) ormyelodysplastic syndrome, cause the emergence of a higherCD86þAS-DCs frequency. This hypothesis can be tested. Myeloiddriver mutation–expressing stem cells, via maturation intoCD86þpDCs, might supposedly engage extrinsic tumor suppres-sion before cell-intrinsic tumor-suppressive signaling cascadessuch as ARF/TP53 can sense oncogenic stress and become acti-vated.Oncogenic stress-sensing tumor-suppressive pathways usu-ally require high-level oncogenic signaling output (56–58), sug-gesting that CML-pDC–induced extrinsic tumor suppression canbe effective long before intrinsic tumor suppression is engaged.

Together, qualitative (pDC-activation and BCR-ABL-status)and quantitative (high versus low pDC counts) factors governthe outcome of pDC biology in CML, which can be tumor-suppressive or -promoting. Further studies are warrantedto eventually exploit oncogene-expressing pDCs as leukemiavaccines.

Disclosure of Potential Conflicts of InterestS. Saussele reports receiving commercial research grant from Novartis and

BMS, and has received honoraria from the speakers' bureau of Novartis, BMS,Incyte, Pfizer, and Roche. A. Hochhaus reports receiving commercial researchgrant from Novartis, BMS, Incyte, and Pfizer. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: S. Inselmann, Y. Wang, M. Huber, A. Hochhaus,A. Burchert

Development of methodology: S. Inselmann, Y. Wang, S. Liebler, T. Ernst,C. Brendel, D. Pavlinic, A. BurchertAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Inselmann, S. Saussele, L. Fritz, C. Sch€utz,M.Huber,T. Ernst, S. Botschek, C. Brendel, D. Pavlinic, V. Benes, A. Hochhaus, A. BurchertAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Inselmann, Y. Wang, S. Saussele, L. Fritz,S. Botschek, R.A. Calogero, E.T. Liu, A. Neubauer, A. Hochhaus, A. BurchertWriting, review, and/or revision of the manuscript: S. Inselmann, S. Saussele,E.T. Liu, A. Neubauer, A. Hochhaus, A. BurchertAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Wang, D. Cai, C. Brendel, V. Benes,A. Neubauer, A. BurchertStudy supervision: A. Hochhaus, A. Burchert

AcknowledgmentsWe thank C. Haferlach (MLL Munich) and D. Haase (University Hospital

G€ottingen) for performingBCR-ABL FISH.We thankC. Fabisch for coordinatingthe CML-V study. We thank Prof. Dr. Zink and Prof. Dr. Engenhart-Cabillic forhelp in performing animal radiation. We thank S. H€uhn for excellent help withpatient sample preparation, cell sorting, and mice work. We thank S. Sopper(University Innsbruck) and D. Wolf (University Bonn) for helpful discussions.

This workwas supported by the "Deutsche Forschungsgemeinschaft "(DFG),Klinische Forschergruppe 210 "Genetics of Drug resistance in Cancer", and theDeutsche Jos�e Carreras Leuk€amiestiftung (AR12/12) and the "Anneliese PohlStiftung." The CML Study V has been supported byNovartis Oncology andMSDSharp & Dohme GmbH. Scientific subprojects related to this study have alsobeen supported by EUTOS.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 13, 2018; revised July 13, 2018; accepted August 27, 2018;published first August 30, 2018.

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Inselmann et al.




2018;78:6223-6234. Published OnlineFirst August 30, 2018.Cancer Res Sabrina Inselmann, Ying Wang, Susanne Saussele, et al. Dendritic Cells in Chronic Myeloid LeukemiaDevelopment, Function, and Clinical Significance of Plasmacytoid

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