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Cancer Therapy: Preclinical

Flt-3L Expansion of Recipient CD8aþ DendriticCells Deletes Alloreactive Donor T Cells andRepresents an Alternative to PosttransplantCyclophosphamide for the Prevention of GVHDKate A. Markey1,2,3, Rachel D. Kuns1, Daniel J. Browne1, Kate H. Gartlan1,3,Renee J. Robb1, J. Paulo Martins1, Andrea S. Henden1,2,3, Simone A. Minnie1,Melody Cheong1, Motoko Koyama1, Mark J. Smyth1,3, Raymond J. Steptoe4,Gabrielle T. Belz5, Thomas Brocker6, Mariapia A. Degli-Esposti7,8,Steven W. Lane1,2,3, and Geoffrey R. Hill1,2

Abstract

Purpose: Allogeneic bone marrow transplantation (BMT) pro-vides curative therapy for leukemia via immunologic graft-versus-leukemia (GVL) effects. In practice, this must be balanced againstlife threatening pathology induced by graft-versus-host disease(GVHD). Recipient dendritic cells (DC) are thought to be impor-tant in the induction of GVL and GVHD.

Experimental Design: We have utilized preclinical modelsof allogeneic BMT to dissect the role and modulation ofrecipient DCs in controlling donor T-cell–mediated GVHDand GVL.

Results:We demonstrate that recipient CD8aþ DCs promoteactivation-induced clonal deletion of allospecific donorT cells after BMT. We compared pretransplant fms-like tyrosine

kinase-3 ligand (Flt-3L) treatment to the current clinical strat-egy of posttransplant cyclophosphamide (PT-Cy) therapy. Ourresults demonstrate superior protection from GVHD with theimmunomodulatory Flt-3L approach, and similar attenuationof GVL responses with both strategies. Strikingly, Flt-3L treat-ment permitted maintenance of the donor polyclonal T-cellpool, where PT-Cy did not.

Conclusions: These data highlight pre-transplant Flt-3Ltherapy as a potent new therapeutic strategy to delete allo-reactive T cells and prevent GVHD, which appears particularlywell suited to haploidentical BMT where the control of infec-tion and the prevention of GVHD are paramount. Clin CancerRes; 24(7); 1604–16. �2018 AACR.

IntroductionUnderstanding the modifiable determinants of alloreactivity

is critical for the design of rational strategies to prevent andtreat graft-versus-host disease (GVHD) after allogeneic bonemarrow transplantation (BMT). The elimination of GVHDmust be balanced against maintenance of protective graft-versus-leukemia (GVL) effects and meaningful separation of

these two immunologic phenomena remains the ultimate goalin the field. Furthermore, retaining functionality within thenonalloreactive T-cell pool is paramount for the prevention ofsevere infection that remains responsible for both short- andlong-term mortality after BMT.

Outside of selecting optimally matched donors, the preven-tion of pathogenic alloreactive T-cell responses (i.e., GVHD)relies on T-cell depletion or pharmacologic immune suppres-sion, usually with calcineurin inhibitors (i.e., cyclosporin ortacrolimus). Posttransplant cyclophosphamide (PT-Cy), whichis thought to eliminate activated, proliferating alloreactivedonor T cells (while sparing regulatory T-cell populations) isalso highly effective in reducing the incidence of chronic GVHDafter haploidentical BMT (1–5).

Retrospective data suggest that PT-Cy may be superior tostandard immune suppression (with or without anti-thymocyteglobulin) with regard to the prevention of chronic GVHD afterunrelated donor transplantation (6) although this requires con-firmation in prospective randomized studies. Interestingly, theeffect of PT-Cy on leukemia relapse is unclear, and importantly,no prospective studies to date have been sufficiently poweredto analyze relapse after BMT using PT-Cy compared with stan-dard immune suppression. With the expanding role of alternatedonor transplantation, it is critically important to understandthe drivers of early T-cell responses that characterize GVL effects

1QIMRBerghoferMedical Research Institute, Brisbane,Australia. 2Royal Brisbaneand Women's Hospital, Brisbane, Australia. 3School of Medicine, University ofQueensland, Herston, Queensland, Australia. 4University of Queensland Dia-mantina Institute, Translational Research Institute, Brisbane, Australia. 5Walterand Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.6Institute for Immunology, Ludwig-Maximilians Universitat, Munich, Germany.7Centre for Experimental Immunology, Lions Eye Institute, Nedlands, WesternAustralia. 8Immunology and Virology Program, Centre for Ophthalmology andVisual Science, University of Western Australia, Crawley, Western Australia.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Kate A. Markey, Bone Marrow Transplantation Labo-ratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia. Phone:617-3362-0290; Fax: 617-3845-3509; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-2148

�2018 American Association for Cancer Research.

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and infectious immunity, such that they may be targeted fortherapeutic benefit.

It is now clear that recipient antigen-presenting cells (APC),both hematopoietic and nonhematopoietic, initiate alloreac-tive donor T-cell responses and GVHD (7), while reconstitutingdonor DCs determine final GVHD severity (8, 9). As yet, nopreventive or therapeutic approaches have been developed tospecifically target the antigen presentation component of T-cellactivation. With regard to GVL responses, recipient APC (puta-tively dendritic cells, but this is as yet unproven) are thought todetermine the magnitude of responses (10), with donor APCplaying a limited role (11).

Given the important effects of CD8þ T cells in HLA-matchedclinical transplantation (12), we have focused on CD8þ T cell-driven mouse models of allogeneic BMT. Here, we have definedthe contribution of dendritic cells (DC) to GVL effects, andexplored the influence of recipient DCs on donor T-cell acti-vation following allogeneic BMT in models that recapitulateboth HLA matched and haploidentical transplantation in theclinic. Surprisingly, we find that recipient CD8aþ DCs potentlyinduce antigen-specific deletion of donor cytotoxic T cells(CTL). We demonstrate that this effect can be exploited toprevent GVHD by further expanding recipient DCs with recom-binant fms-like tyrosine kinase-3 ligand (Flt-3L); notably, thisstrategy appears superior to current approaches for in vivo T-celldepletion using PT-Cy, as the GVHD outcome is superior andthe polyclonal T-cell pool appears maintained.

MethodsMice

Female C57BL/6 (B6.WT, H-2b), Ptprca (B6.Ptprca, H-2b,CD45.1; also described as B6.CD45.1 for clarity), and B6D2F1(H-2b/d) mice were purchased from the Animal Resources Centre(WA, Australia). The following strains were bred and housed atQIMRB: C3H.Sw (H-2b, Ly9.1), B6.CD11c.DOG (H-2b) and B6.CD11c.DOGxDBA/2F1 (H-2Db/d; diphtheria toxin (DT) receptor,ovalbumin (OVA), and enhanced GFP (eGFP) driven off theCD11c promoter), B6.CD11cGCDL (eGFP, Cre, the DTR, lucif-erase driven off the CD11c promoter), B6.IL12p40 eYFP (13),OT-I Tg, CD11c.Rac Tg (14, 15), IRF8�/� mice [>10 x backcrossesto B6 background, lacking in CD8aþ DC; ref. 16), Batf3�/�

(lacking in CD8aþ DC, (17)], b2m�/�, Bm1 (H-2Kbm1), andBm1.ActmOVA (H-2Kbm1 and ubiquitous ovalbumin expressiondriven off the b-actin promoter). Female mice at 8 to 12 weeks ofage were used throughout the study.

Bone marrow chimerasFor IRF8�/� chimeras, bone marrow was harvested from

IRF8�/� mice and 5 � 106 bone marrow cells/mouse transferredby intravenous injection into lethally irradiated (1,000 cGy) B6.WT or B6.CD45.1 (PTprca) recipients and allowed to reconstitutefor 3 months prior to second transplantation.

Bone marrow transplantation, leukemia induction, andtreatment schedules

B6 recipients of C3HSw grafts received 1� 106 FACS purified(CD90.2þ/CD4�) CD8þ T cells and 5 � 106 bone marrow cells.Where T cell depleted (TCD) bone marrow controls wereincluded, cells were prepared using an antibody incubationfollowed by complement depletion as described previously(18). In the C3H.Sw model, donor mice were immunized viaintraperitoneal injection with B6 splenocytes 2 weeks prior totransplantation. B6D2F1 recipients received 5 � 106 TCD bonemarrow cells þ either magnetic bead–purified CD8þ T cells(MACS-purified according to the manufacturer's instructions,Miltenyi Biotec), CD3þ T cells (as previously described; ref. 18),or OT-I Tg CD8þ T cells in doses as stated. OT-I were MACS-purified from spleen and lymph nodes. Where OT-I T cells weretransferred to read out antigen-specific responses, MHC classI–deficient (b2m�/�) bone marrow was used to exclude anycontribution of indirect antigen presentation by donor APC.

Total body irradiation (TBI) doses were as follows: B6 back-ground, 900 cGy in the presence of DT treatment and 1,000 cGyotherwise; B6D2F1 mice, 1,100 cGy; Balb/B mice 400 cGy (inconjunction with 2 mg fludarabine d-4 to d-2).

For in vivo depletion of DTR-expressing DCs, diphtheriatoxin from Corynebacterium diphtheriae (Sigma Aldrich) wasadministered intraperitoneally at 160 ng per dose on day�2, �1, 0, 1, and 2 for day 3 analyses and continued on day5, 7, 9, for day 10 analyses. Primary leukemia cells weregenerated using the expression of the human oncogenesMLL-AF9 or BCR-ABL þNUP98-HOXA9 to model human AMLand myeloid blast-crisis leukemia, as described previously (19)and cryopreserved at disease onset, for subsequent transplan-tation. Leukemia cells were thawed on the day of injection andincluded in grafts at 0.5–1 � 106/mouse. Mice were scoredaccording to standard protocols and sacrificed if clinical scorereached �6, in accordance with animal ethics guidelines and apreviously established scoring system (20). For a death to beattributed to leukemia, leukemia burden in peripheral bloodat either terminal or last routine bleed had to meet the fol-lowing criteria to avoid overstatement of leukemic deaths forthe rare cases when both GVHD and leukemia were present:greater than 10%GFPþ cells in peripheral blood (with any totalwhite cell count) or present in the peripheral blood at any levelbut with a total WCC � 1 � 107/mL.

For in vivo expansion of DCs, mice received 10 mg of Flt-3ligand, daily via subcutaneous injection (Flt-3L; CelldexTherapeutics) from d-10 to -1 (21, 22). Posttransplant cyclo-phosphamide (PT-Cy) was administered on dþ3 and dþ4 at100 mg/kg i.p. Cyclosporin was administered at 5 mg/kg i.p.from d0 to dþ14.

Translational Relevance

Graft-versus-host disease (GVHD) is a major barrier tosuccessful allogeneic bone marrow transplantation, a proce-dure offering curative potential to patients with hematologicmalignancies and marrow failure syndromes. In our preclin-ical models, pretreatment with Flt-3L (pre-T Flt-3L) expandsrecipient CD8aþ DCs that subsequently activate and deleteantigen-specific donor T cells, an effect similar to that seenwithposttransplant cyclophosphamide (PT-Cy). Pre-T Flt-3L offersprotection from GVHD while facilitating the maintenance ofthird-party reactive donor T cells. Pre-T Flt-3L, like PT-Cy,resulted inmarked reductions in graft-versus-leukemia effects.As Flt-3L has shown acceptable safety profiles in phase I/IIclinical trials, this approach to prevent GVHD is readilytestable.

Recipient DCs Drive Deletion of Donor Ag-specific T Cells

www.aacrjournals.org Clin Cancer Res; 24(7) April 1, 2018 1605




For poly I:C experiments, mice received 100 mg of poly I:C(Invivogen) in saline via intraperitoneal injection 1 hour fol-lowing the second dose of TBI, and prior to the injection of OT-IT cells.

CMV studiesFemale C57BL/6 mice were infected intraperitoneally

with 104 PFU of salivary gland propagated MCMV-K181-Perthfor >90 days to establish a latent MCMV infection, as deter-mined by the absence of replicating virus. Splenic T cells wereisolated from latently infected mice and transplanted usingthe same methods described below. MCMV-infected micewere housed at the University of Western Australia prior tobeing used as BMT donors. CMV-specific CD8þ T cells wereidentified using PE-conjugated tetramer for H-2Kb-SSPPMFRVMCMV-m38 (ImmunoID Tetramers, University of Melbourne,Australia).

Gene expressionTotal RNA was extracted with the RNeasy Mini Plus kit (Qia-

gen) from sort-purified (>95% purity) CD8þ donor-type T cellsand gene expression measured using the Qiagen RT2 Profiler PCRArrayMouse Apoptosis kit (Qiagen), and validated using TaqManGE assays (Applied Biosystems).

Xenogen imagingBioluminescent imaging was performed to demonstrate the

depletion and expansion of DCs (using B6.CD11c.GCDL mice,and chimeras in which the hematopoietic compartment alonewas of B6.CD11cGCDL origin). Recipients were injected subcu-taneously (SC) with D-Luciferin (0.5 mg, PerkinElmer) and thenanesthetized with isoflurane 5 minutes before imaging using theXenogen imaging system (Xenogen IVIS 100; Caliper LifeSciences; ref. 23). Bioluminescence (BLI) shown as photons persecond (ph/s).

In vivo and in vitro cytotoxicity assaysIn vivo cytotoxicity assays were performed as follows: 10

days after BMT, recipient mice received 2 � 107 congenicdonor-type (PTprca, CD45.1þ) unlabeled splenocytes and2 � 107 host-type B6D2F1 CD45.2þ CFSE-labeled splenocytesIV (24). Eighteen hours later, peripheral blood and spleenwere analyzed for remaining donor (CD45.1þ PE-stained) andhost-type (CFSE-labeled) cells by FACS analysis. The ratio ofadoptively transferred donor to recipient cells in spleen after18 hours reflects degree of cytotoxicity of the resident donorT-cell population and is reported as: n donor-type/n host-typecells recovered.

For in vitro cytotoxicity, allogeneic (B6D2F1 BCR-ABL þNUP98-HOXA9) and syngeneic (B6 MLL-AF9) primary leukemiatarget cells were labeled with 51Cr, prior to culture with donorCD8þ effector T cells (FACSpurified from recipient spleens onday10 following BMT, CD45.1þ/CD8þ) for 5 hours at 37�C, 5%CO2.51Cr release into culture supernatant was determined via gammacounter (TopCount microplate scintillation counter, PackardInstruments). Spontaneous release was determined using wellscontaining labeled target only, and maximum release from wellscontaining targets þ 1% Triton X�100. Percentage cytotoxicitywas determined as follows: percentage cytotoxicity¼ (experimen-tal release � spontaneous release)/(maximum release � sponta-neous release) � 100.

Flow cytometryA full list of mAbs utilized is given in Supplementary

Table S2. Annexin V was assessed as previously described(18). Intracellular staining with activated caspase-3 was per-formed according to the manufacturer's instructions (BD Phar-mingen). Flow cytometry analysis was performed using anLSR Fortessa II (BD Biosciences) using FACSDiva software(Version 8.0.1). Offline analysis was performed using FlowJo(Version 10, Treestar).

Statistical analysisSurvival curves were plotted using Kaplan–Meier estimates

and compared by log-rank analysis. Unpaired two-tailedMann–Whitney tests were used throughout. Data are mean� SEM and P < 0.05 considered significant. GraphPad Prism(Version 6.00 for Windows, GraphPad Software, www.graphpad.com) was used for the generation of graphs and forstatistical analysis. Researchers were not blind to the groupsat the time of analysis. �, P � 0.05; ��, P � 0.01; ��, P � 0.001;��, P � 0.0001. Sample sizes for mouse experimentswere estimated on the basis of our expected effect size andprevious experience with these models. Where all recipientmice were WT, formal randomization was not conducted,but groups were matched for weight at the commencementof experiments.

ResultsRecipient DCs regulate GVL effects

Immune-mediated antitumor effects are increasingly recog-nized as central to long-term disease control following clin-ical transplantation. The contribution of recipient DC toCD8þ T-cell–mediated GVL effects was examined in a CD8þ

T-cell–dependent, miHA mismatched C3H.Sw ! B6 mousemodel of BMT (25). Recipient B6.CD11c.DOG mice (wherediphtheria toxin receptor, OVA, andGFP are driven off the CD11cpromoter) with either DCs intact (saline treated) or DCs depleted(diphtheria toxin treated) were transplanted with C3H.Sw bonemarrow and T cells, as well as B6-derived (recipient-type) MLL-AF9–transduced leukemia, to mimic the clinical potential forrelapse in parallel with the immunologic GVL effect exerted bythe donor graft (19, 26). Unexpectedly, mice developed higherleukemia burdens and died more rapidly when recipient DCswere present compared with when they had been depleted,suggesting that DCs somehow function to attenuate GVL effects(Fig. 1A and B). This model does not induce GVHD mortalityunder control conditions, and as such, survival data reflects solelyleukemia-related death. Of note, when we performed theseexperiments using a lymphoma cell line (EL4, which generateshepatosplenic masses rather than a leukemic phase) to modelGVL there were no differences between DC-depleted and -repleterecipients (data not shown).

As CD8aþ DCs have been implicated in tolerance induction(27, 28), we next performed transplants using recipients lackingIFN regulatory factor 8 (Irf8) gene expression in the hematopoieticcompartment (Irf8�/�! B6 chimeras), and thus missing CD8aþ

DC (Fig. 1C). The improvement in survival in the isolated absenceof CD8aþ DC mimicked that observed in the setting of pan-DCdepletion (Fig. 1D and E), suggesting CD8aþ DCs are the keypopulation required for control of leukemia relapse. RecipientDCs as a whole, and the CD8aþ DC subset specifically are lost

Markey et al.

Clin Cancer Res; 24(7) April 1, 2018 Clinical Cancer Research1606



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rapidly after total body irradiation (TBI), and as expected, theCD8aþ DCs preferentially produce IL12, both homeostaticallyand following TBI (Fig. 1F and G).

CD8aþ DCs are known to be highly efficient at the uptakeof cell-associated antigen from dying cells (29, 30); we there-fore investigated the contribution of this pathway (i.e., cross-presentation) to leukemia control after allogeneic BMT. Weutilized Rac1 transgenic recipients, where DCs specifically lackthe capacity to acquire apoptotic antigen due to deficiency ofthe Rho GTP-ase Rac1 within CD11cþ cells (14). Surprisingly,there was no impairment in GVL (Fig. 1H), suggesting thatrecipient DCs regulate GVL effects via the presentation ofendogenous alloantigen, rather than cross-presentation ofexogenous alloantigen.

Recipient DCs induce apoptosis in donor T cells, which resultsin contraction of the polyclonal T-cell compartment

To understand the profound modulation of CD8þ T cell-mediated GVL effects by recipient DCs, we examined the donorT-cell compartment after BMT. Donor CD8þ T-cell numberswere equivalent following transplantation into DC-depleted orDC-intact mice at d3, but were significantly reduced by d7 inrecipients with DCs intact (Fig. 2A). This was associated with amarked increase in donor cells undergoing apoptosis at bothtime points examined (Fig. 2B). The same effect was observed inboth IFR8�/� ! B6 chimeras and Batf3�/� recipients (17) thatlack the CD8aþ DC subset in isolation (Fig. 2C and D).

We next analyzed donor CD8þ T cells sort-purified from DCdepleted or intact recipients on d7 using a targeted PCR array.

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Figure 1.

IL12-producing recipient CD8aþ DCs accelerate leukemia relapse. A, B6.CD11c.DOG or B6.WT mice were irradiated (900 cGy, d0) and transplanted with1 � 106 sort-purified CD8þ T cells and 5 � 106 bone marrow cells, or TCD bone marrow alone, from C3H.Sw donors, with 1 � 106 GFPþ MLL-AF9primary mouse leukemia cells. Mice were treated with saline (DC intact) or DT (DC depleted) from day �2 to þ7. Mice were bled weekly andsamples were counted and lysed prior to analysis via FACS for presence of GFPþ cells. Data represent three replicate experiments, d7 n ¼ 5/group; d14and 21, n ¼ 15/group TCD mice, n ¼ 17–18/group BM þ T mice; d28 n ¼ 2 TCD mice (only living mice at this time point from a large number ofreplicate mice), n ¼ 13–16/group BM þ T mice. Mean � SEM shown. d28 DCs depleted versus DCs intact; P ¼ 0.0007. B, Kaplan–Meier curveshowing time to death from leukemia for two replicate experiments as described in A. Total n ¼ 10 (TCD group); 15 (DC intact BM þT); 16 (DC depletedBMþT). TCD versus BMþT with DC intact, P < 0.0001; BMþT DC intact versus DC depleted, P < 0.0001. C, Splenic DC subsets in WT [B6.CD45.1 ! B6]chimeras and [IRF8�/� ! B6] chimeras. D, Leukemia engraftment of IRF8�/� chimeric mice receiving C3H.Sw grafts as described. E, Kaplan–Meiercurve showing time to death from leukemia for two replicate experiments. n ¼ 7–9 in TCD groups, n ¼ 17 in each BMþT group. F, SplenicDC were enumerated in from WT B6 mice at rest, and 18 hours following TBI (1,100 cGy). n ¼ 9 for control mice, 16 for TBI group. Data shown fromthree replicate experiments. G, IL12 production was measured using IL12 eYFP reporter mice, as described in F. n ¼ 9 for control animals, 8 for post-TBI.H, CD11c.Rac1 Tg recipient mice were transplanted as described in A. Kaplan–Meier curve for leukemia death from two replicate experiments, n ¼ 10/TCDgroup, n ¼ 16/BMþT groups.






Figure 2.

Recipient DCs induce apoptosis in polyclonal donor CD8þ T cells. A, Recipient B6.CD11c.DOG mice were treated with DT or saline and transplanted with bonemarrow and sort-purified CD8þ T cells from C3H.Sw donors. Splenic donor CD8þ T cells were analyzed at day 3 and 7, with enumeration from tworeplicate experiments shown. n¼ 9/group at d3 and 10/group at d7. B, Proportion of cells undergoing apoptosis, defined by Annexin Vþ/7AAD� shown in left.Representative FACS plots in right panel. Data shown from two replicate experiments. n ¼ 5/group at d3, 10/group at d7. IRF8�/� chimeras (C) and Batf3�/�

mice were used as recipients (D). Cells were phenotyped using flow cytometry. Enumeration was performed on the basis of whole-organ WBC(established by Coulter count) and phenotype and data from day 3 and 7 is shown. Data represent two replicate experiments for each. n ¼ 5/group atd3, 10/group at d7 for the [IRF8�/� ! B6] and n ¼ 8/group/time point in the Batf3�/� experiments. E, Apoptosis gene array on sort-purified donorT cells (CD8þ/Ly9.1þ from the C3H.Sw ! B6.CD11c.DOG � DT transplant model. Differentially regulated genes (Igfr1, Trp63, Trp73, and DapK1) shownin red. Array performed on individual sorted T cells from day 7 posttransplant, n ¼ 4/group. F, TaqMan real-time qPCR results for d10 ex vivo T cells fromthe B6 ! B6.CD11c.DOG � DBA/2F1 � DT system. qPCR-sorted splenic T cells from individual mice, n ¼ 4/group.

Markey et al.





We identified four genes that were expressed at higher levels inthe T cells from mice that had not been exposed to recipientDCs, all of which are involved in regulation of cellular growthand apoptosis: Insulin-like growth factor 1 receptor (Igfr1; 4.44fold), transformation-related protein 63 and 73 (Trp63; 2.29fold and Trp73; 3.00 fold), and death associated protein kinase1 (DapK1; 2.22 fold; Fig. 2E). This likely reflects the loss of,or lack of requirement for, regulation among the nonalloreac-tive pool of T cells that remain post-DC-mediated deletion.These changes in gene expression were validated by qPCR in thehaploidentical model at d10, with consistent results obtained(Fig. 2F). The full panel of genes analyzed is listed in Supple-mentary Table S1.

Recipient DC:CD8þ T-cell interactions result inantigen-specific deletion and loss of cytolytic function

We next measured antigen-specific T-cell functionusing CD8þ OT-I transgenic donor T cells and B6.CD11c.DOGxDBA/2 F1 recipients, with or without DT treatment todeplete DCs. Ovalbumin (OVA), which is expressed by recip-ient DC (driven by the CD11c promoter), serves as a modelalloantigen in this system. OT-I T cells exclusively recognize theOVA-derived peptide SIINFEKL in the context of MHC class I,and therefore OT-I responses quantify DC-specific, endogenouspresentation of OVA-derived peptide. Once again, we observedDC-associated compartment size contraction and enhancedapoptosis (Fig. 3A and B), as well as hyperactivation of OT-Itransgenic T cells (CD25 and CD69 expression, Fig. 3C). OT-Iacquired an effector phenotype (CD62Lneg/CD44high) morerapidly in the presence of DCs (78.0 � 0.9% in DC repletevs. 33.2 � 1.8% in DC deplete at 3 days after BMT; Fig. 3D).Strikingly, recipient DCs induced high levels of exhaustionmarkers PD-1 and Lag3 on T cells early after BMT, and Tim3was present at high levels, but was not differentially expressed(Fig. 3E). Exhaustion marker expression was markedlydecreased in both T-cell groups by d10, with enhanced expres-sion seen in the T cells from DC-depleted recipients likelyreflecting the delayed, more measured activation that occurswhen initial interaction with DC is denied.

To assess the impact of DCs on antigen-specific T cells withina polyclonal pool (containing both CD4þ and CD8þ T cells),we used the parent-into-F1 (B6 ! B6D2F1) model of haploi-dentical transplantation and waited until donor T-cell numberswere equal between DC-depleted and -replete recipients (d10,Fig. 3F). We noted reduced cytotoxic function in vivo (Fig. 3G),and a striking absence of in vitro cytotoxic function againstrecipient-type leukemia (Fig. 3H), consistent with the specificdeletion of alloreactive CTL, even when a full complement ofpolyclonal T cells are present in the initial graft.

Expansion of recipient DCs with Flt-3L results in an earlyexpansion of antigen-specific T cells followed by their completedeletion in lymphoid organs

Having established recipient CD8aþ DCs as key drivers ofalloantigen-specific donor T-cell deletion, we sought to furtherharness this effect by administering Flt-3L pretransplantation(Pre-T Flt-3L). Expansion of CD8aþ DC was confirmed bybioluminescence and flow cytometry (Fig. 4A and B). WhenOT-I T cells were transferred into Pre-T Flt-3L conditionedB6.CD11c.DOGxDBA/2F1 recipients, OT-I were present inincreased numbers in spleen and lymph nodes 12 hours after

transfer when compared with saline-treated controls (Fig. 4Cand D). Strikingly, by day 3, the reverse was true, with OT-Iundetectable in Flt3-L–treated animals, consistent withthe induction of widespread deletion following initial stimu-lation and early expansion (Fig. 4E). By day 10, splenic OT-Inumbers were equivalent in the Flt-3L and saline groups,suggesting a reexpansion of surviving T cells in the Flt-3Lpretreated mice. These were, however, markedly differentto the OT-I present in saline pretreated mice, skewed towarda TEM/TEFF phenotype and a high proportion were undergoingactive apoptosis, as measured by activated caspase-3 intracel-lular staining (Fig. 4F).

Flt-3L–expanded DCs specifically delete alloreactive T cellswhile maintaining the polyclonal T-cell pool

To examine the functional consequences of Flt-3L expansionon alloreactivity, we first enumerated polyclonal B6 T cells(both CD4þ and CD8þ) following transplantation into salineor Flt-3L–treated B6D2F1 recipients, a model of haploidenticaltransplantation in the clinic. Interestingly, polyclonal T cellswere preserved in the setting of Flt3L treatment (Fig. 5A). Toexplore the role of antigen-specific T-cell deletion in leukemiarelapse, Pre-T Flt-3L recipient mice were transplanted withBCR-ABL þ NUP98-HOXA9 cotransduced primary leukemiawith bone marrow � polyclonal CD3 T cells (31, 32). Medianleukemia survival was just 11 days in the Pre-T Flt-3L BM þ Tgroup, equivalent to recipients of T-cell–depleted (TCD) con-trol grafts. The saline pretreated mice receiving bone marrow þT grafts survived long-term (Fig. 5B and C).

We confirmed that this was a DC-specific effect by using Flt-3Lpretreatment in combination with the DT-depletion approachpreviously described (Fig. 5D and E). Themice treated with Flt-3LandDT (such that DCs were expanded and subsequently deleted)had equivalent leukemia control to that seen in the WT bonemarrowþ T recipients pretreatedwith saline (Fig. 5E), confirmingthe primacy of recipient DCs in this effect.

To assess whether this deletion was due to the potentiallytolerogenic nature of CD8aþ DCs, we treated mice with Flt-3Lor saline as described, and then activated recipient DCs withtoll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylicacid (poly I:C) prior to the infusion of antigen-specific OT-I Tcells. As expected, DCs were highly activated following poly I:C treatment (Fig. 5F; ref. 33). Importantly, complete deletionof antigen-specific T cells in Flt-3L pretreated recipients wasmaintained in the setting of this DC activation (Fig. 5Gand H). Interestingly, while deletion was most effective inthe Flt-3L pretreated mice, poly I:C activation of residualDCs also lead to marked donor T-cell depletion in salinepretreated recipients. Thus, the ability of recipient DCs toattenuate GVHD reflects direct alloantigen presentation andtheir capacity as highly potent APC rather than any intrinsicregulatory property.

We next assessed the impact of posttransplant cyclophos-phamide (PT-Cy) on antigen-specific T-cell depletion, main-tenance of the polyclonal T-cell pool and ultimately, GVLeffects. As expected, the high-dose chemotherapy effectivelyeliminated the alloreactive OT-I T cells (Fig. 5I), but also hadsignificant impact on the polyclonal T-cell pool when WT B6grafts were transplanted (Fig. 5J). PT-Cy had a significantimpact on leukemia relapse. While the PT-Cy TCD groupexperienced delayed relapse compared with saline-treated






animals (median survival 22 days compared with 14.5), likelydue to direct effects of the cyclophosphamide on the leukemiacells. PT-Cy resulted in rapid relapse in the bone marrow þ Trecipients (median survival 26 days, compared with unreach-ed in the saline-treated controls, as no mice died of leukemia;Fig. 5K).

Following on from this striking and rapid relapse in thesetting of PT-Cy, we next performed experiments using clini-cally relevant calcineurin inhibition (CsA, 5 mg/kg, day 0–14,achieving trough levels between 200 and 300 mg/dL; Fig. 5L).

CsA-treated mice relapsed in an equivalent fashion to the salinetreated controls, and the PT-Cy mice once again relapsed atsimilar rates whether T cells were present in the graft, or graftswere TCD marrow alone.

Flt-3L pretreatment is a superior strategy for prevention ofGVHD when compared with PT-Cy

Until there are clear therapeutic strategies which separateGVHD and GVL, it is likely that any strategy chosen to preventand treat GVHD will have some influence on relapse risk. In

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Figure 3.

Donor T-cell deletion is antigen-specific. A, Recipient B6.CD11c.DOG � DBA/2 F1 mice were treated with DT or saline from d�2 to þ9, received TBI on d0and were transplanted with 1 � 106 OT-I T cells and 5 � 106 b2m�/� BM. d3 and d10 enumeration. Four replicate experiments, n ¼ 7/group at d3; 8, 9, 17respectively at d10. B, Proportion of d10 OT-I T cells undergoing apoptosis, n ¼ 7/group from two replicate experiments. C, Plots show splenic activationmarker expression by OT-I T cells on d10. n ¼ 4/group, representative data from one of two replicate experiments. D, Effector/memory phenotype;representative flow cytometry plots and enumeration. Data shown are representative of three replicate experiments. n ¼ 5/group at d3, n ¼ 10/group atd10. E, Exhaustion markers at day 3 and 10; MFI shown for Lag3, PD-1, Tim3. Data representative of three replicate experiments. Data shown, n ¼ 5/groupfor PD-1 and 10/group for Lag3 and Tim3. F, Polyclonal B6 CD8þ T cells were transplanted into B6.CD11c.DOG � DBA/2F1 recipients. T cells wereenumerated at day 10. Data shown from four replicate experiments, n ¼ 17/group. G, In vivo cytotoxicity assays were performed as described, cytotoxicityindex is reported, with data shown combined from three experiments, n ¼ 14–15/group. H, Polyclonal donor CD8þ T cells were sort-purified onday 10 post-BMT and plated with allogeneic (B6D2F1 BCR-ABL þ NUP98-HOXA9) and syngeneic (B6 MLL-AF9) primary leukemias in a chromiumrelease assay. Data shown is one of two experiments performed with equivalent results. Each replicate experiment, n ¼ 5/group (T cells purifiedfrom individual mice).

Markey et al.





that setting, we sought to assess the relative benefit from aGVHD point of view from PT-Cy and Flt-3L pretreatment,given that both have a detrimental (and equivalent) impacton leukemia relapse. We found that Flt-3L pretreatment was

superior to PT-Cy (median survival 45 vs. 36 days) forGVHD prevention (Fig. 6A) in a haploidentical model. Giventhat Flt-3L is a myeloid growth factor, efforts to translatethis strategy for GVHD prevention would need to begin in

A B

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Figure 4.

Flt-3L pretreatment results in early antigen-specific T-cell expansion followed by near-complete deletion. A, B6.CD11c.GCDL mice (luciferase driven offthe CD11c promoter) were pretreated with Flt-3L for 10 days and imaged using the Xenogen IVIS. Representative images are shown of 5 mice/group.Quantification is demonstrated for whole body, n ¼ 5/group. B, Flow cytometry plots demonstrating DC identification and subset, n ¼ 5/group. C, OT-I T cellswere transferred into irradiated B6.CD11c.DOG � DBA/2 F1 recipients pretreated with either Flt-3L or saline for 10 days. Mesenteric lymph node (mLN),peripheral lymph node (pLN) and spleen shown, plots are concatenated and representative of 4 individual animals harvested at 12 hours post-transfer.Enumeration shown in D. E, Representative flow plots of spleen at day 3. Data pooled from two replicate experiments (n ¼ 10/group). OT-I T cellswere enumerated in spleen, at day 3 and day 10. F, T-cell phenotype at day 10 from experiment as described in C. Donor OT-I T cells were identified onthe basis of their TCR expression (Va2/Vb5.1) and phenotyped on the basis of CD62L and CD44 expression. Apoptosis was assessed using intracellularstaining for activated caspase-3. Data pooled from two replicate experiments n ¼ 6–7/group.






the lymphoid malignancies, in which setting patients are oftenconditioning with reduced intensity regimens. We thereforeperformed transplants in an additional miHA mismatch mod-el (B6 to Balb/B) with fludarabine and low-dose TBI condi-tioning. The benefit of Flt3L pretreatment was maintained in

this setting, with no GVHD deaths in the Flt-3L pretreatedrecipients of bone marrow þ T grafts (Fig. 6B), and equivalentengraftment.

Having observed profound effects on broad alloreactivity (i.e.,bothGVL andGVHD) in response to both Pre-T Flt-3L and PT-Cy,

A

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Figure 5.

Flt-3L pretreatment results in functional deletion of the alloreactive T-cell pool with maintenance of the polyclonal T-cell pool. A, CD3þ T cells from B6 donorswere transplanted into B6D2F1 mice pretreated with Flt-3L or saline as described. Enumeration of polyclonal splenic T cells on d3 is shown. Data arefrom two replicate experiments, n ¼ 8/group. B, B6D2F1 mice were treated with either Flt-3L or saline for 10 days prior to TBI and transplantation of B6.WT BM � 0.5 � 106 CD3þ T with BCR-ABL þ NUP98-HOXA9 leukemia cells on day 0. Kaplan–Meier survival curve shown. Data shown are combinedfrom two replicate experiments. n ¼ 9/TCD BM group, n ¼ 14/BMþT group. C, Leukemia burden at d10 is shown, from the two combined experimentsdescribed. BMþ T grafts, saline versus Flt-3L, P < 0.0001. D, B6D2F1 mice were treated with Flt-3L and either saline or DT. Representative FACS plots pre- andpost-TBI shown. Plots shown are gated on FSC/SSC, 7AAD-negative, single cells. Axes for each plot are as shown. E, Mice were transplanted followingtreatment described in D. Data shown is from two combined replicate experiments. n ¼ 9 in TCD group; 12 in saline controls, 16 in Flt-3L � DT depletion.F, Recipient dendritic cell activation status 18 hours following Poly I:C treatment (100 mg, i.p.). Gated on CD11cþ/MHC class IIþ cDC. G, Mice werepretreated with Flt-3L as described, and additionally treated with either Poly I:C or saline post-TBI and prior to the injection of 1 � 106 OT-I T cells.Representative FACS plots shown in G are gated on FSC/SSC, 7AAD-negative, single cells. Enumeration of splenic OT-I cells in spleen on day 3 is shownin H with data pooled from two replicate experiments, n ¼ 8/group. I, Antigen-specific OT-I CD8 T cells were transplanted as previously describedinto irradiated B6.CD11c.DOGxDBA/2F1 recipients. Mice were treated with saline or cyclophosphamide on day 3 and 4 posttransplant, and spleensanalyzed on day 7. Data shown from two replicate experiments, n ¼ 13 (saline) and n ¼ 5 (PT-Cy). Representative flow cytometry plots are shown,gated on FSC/SSC, 7AAD-negative, single cells. OT-I T cells are defined on the basis of TCR expression (Va2/Vb5.1). J, CD3þ T cells from B6 donorswere transplanted into B6D2F1 mice and treated with PT-Cy as described. Enumeration of polyclonal splenic T cells on d7 is shown. Data shown isrepresentative of two replicate experiments, n ¼ 4/group. K, B6D2F1 mice were transplanted as described above, and treated with saline or 100 mg/kgcyclophosphamide on d3 and d4. Kaplan–Meier curve for leukemia death. Data shown is pooled from three replicate experiments, n ¼ 14 in saline andCy treated TCD; n ¼ 22 in the BMþT. L, B6D2F1 mice were transplanted as described, with grafts containing BCR-ABL þ NUP98-HOXA9 primary leukemia,but treated with either PT-Cy or CsA (5 mg/kg/day for day 1–14). Kaplan–Meier curve shown for leukemia death. Data are pooled from two replicateexperiments, n ¼ 7 in each TCD group and 11 in each BMþT group.

Markey et al.





we sought to examine maintenance of virus-specific immunityusing murine cytomegalovirus (MCMV) immune B6 donor micein the haploidentical B6! B6D2F1model.We hypothesized thatin the absence of CMV-specific antigen in the peritransplantperiod, the CMV-specific memory T-cell pool would be intactfollowing transplant into Flt-3L-pretreated recipients. At the timeof maximal clonal deletion of alloreactive T cells, CMV-specific(m38 tetramerþ cells) were indeed present in equivalent numbersin Flt-3L pretreatedmice comparedwith saline pretreated controls(Fig. 6C). In contrast, there was profound reduction in all Tlymphocytes in the PT-Cy mice, with almost complete loss ofCMV-specific CD8 T cells (Fig. 6D). Thus unlike PT-Cy, pre-T Flt-3L preserves virus-specific T cells.

DiscussionIn this study, we have examined the role of recipient DCs in

controlling CD8þ T-cell–mediated alloreactivity and proposethat pretransplant Flt-3L therapy may provide a new strategyfor the prevention of GVHD in the clinic. It is clear that thiscomes at the cost of decreasing T-cell–mediated GVL effects,

but in the era of posttransplantation cellular therapies (e.g.,with engineered CAR T cells or suicide-gene transfected poly-clonal donor T cells) it is likely that this can be overcome.Importantly, pretransplant Flt-3L therapy appears to spare thepolyclonal T-cell pool and in light of the importance of oppor-tunistic infection after transplant, this represents a significantadvantage over nonselective chemotherapy-based approachesfor the deletion of alloreactive T-cell populations that arecurrently used. In addition to the polyclonal T-cell pool, Flt-3L appears to spare CMV-specific immunity where PT-Cy doesnot. This is likely explained by the relative absence of viralantigen in the immediate post-transplant period.

Mechanistically, we demonstrate that alloantigen presenta-tion within MHC class I by recipient CD8aþ DC results inactivation induced cell death and exhaustion of allospecificCD8þ cytolytic T cells. We thus demonstrate the ability of DCsto delete MHC class I–dependent GVL effects, as well as CD8þ

T-cell–mediated GVHD. A previous study using Batf3-deficientrecipients and a T-cell lymphoma cell line to model relapseafter BMT reported that CD8aþ DCs were required for optimalGVL effects, which is in contrast to our findings using primary

Day 3

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Figure 6.

Flt-3L treatment results in superior GVHD prevention when compared with PT-Cy. A, B6D2F1 mice were pretreated with either Flt-3L or saline, transplantedwith B6 BM � 5 � 106 CD3þ T cells, and administered posttransplant cyclophosphamide or saline on dþ3, dþ4. Kaplan–Meier survival curve shown.Pooled data from three replicate experiments, n ¼ 15/group. B, Balb/B mice were conditioning with fludarabine (2 mg/dose d�3, �2, �1) and sublethalirradiation (400 cGy, d�1), followed by transplantation with B6 grafts (1 � 107 BM � CD3þ T cells). Kaplan–Meier survival curve shown, pooled fromthree replicate experiments. n ¼ 10 in the TCD arm and 18 in each of the BM þ T arms. C, 5 � 106 CD3þ T cells from MCMV immune donors were transplantedinto B6D2F1 mice pre-treated with Flt-3L or saline as described. Splenic T cells, including MCMV-specific m38þ CD8þ T cells, were examined 3 days later;enumeration is shown in the right panels. FACS data shown is representative of two replicate experiments. Enumeration data is pooled n ¼ 8/group. D, Micewere transplanted as described, but treated with cyclophosphamide on dþ3 and dþ4 and splenic T cells, including MCMV-specific m38þ CD8þ T cellsexamined 3 days later at dþ7 (n ¼ 4/group).






myeloid leukemias (34). Indeed, we could see no effect ofrecipient DC depletion on GVL against the EL4 cell line (datanot shown). This likely reflects the high mutational burdens inthe multiply passaged cell lines used in those studies relativeto primary leukemia (35) and the fact that these models arepoor discriminators of quantitative defects in GVL (12). Theresults presented here also serve to confirm and extend datafrom ourselves and others (7, 36, 37) demonstrating thatrecipient DCs are not required for the induction of GVHD.Indeed, we have previously shown that recipient DCs alsopotently delete antigen-specific donor CD4 T cells to attenuateGVHD (7).

Our work demonstrates that DCs act to control alloreactivityvia their profound stimulatory capacity, which results in clonalT-cell deletion. This effect can be augmented by expandingrecipient CD8aþ DCs (e.g., with Flt-3L), or enhancing residualDC activation (e.g., with poly I:C). Interestingly, the adminis-tration of exogenous IL12 within 12 hours of BMT has beenshown to prevent GVHD by inducing donor T-cell apoptosisand is again consistent with the CD8aþ DC–mediated effectdemonstrated here (38–40). While the administration of IL12has significant potential for toxicity in clinical BMT and has notprogressed into the clinic, Flt-3L has been widely administeredto patients (41, 42) and does represent a feasible clinicalapproach.

When Flt-3L therapy was previously studied in GVHD, itappeared to have mixed effects, dependent on the timing ofadministration (21, 43). Flt-3L administration post-BMT expan-ded donor DCs and resulted in the expected acceleration ofGVHD (43). Conversely, treatment of recipients prior to BMTappeared to reduce GVHD, and the authors observed lowernumbers of donor T cells early after transplantation. At thattime, this was interpreted as a failure of Flt-3L–expanded DCs tostimulate T-cell expansion, as DCs were thought to have anexclusively stimulatory function (21).

Expansion of CD8aþ DC using Pre-T Flt-3L represents apotent immunomodulatory strategy that appears to be at leastequivalent to high-dose PT-Cy for the deletion of alloreactive Tcells using our haploidentical transplant model. While PT-Cyhas been shown to spare regulatory T cells (4), it is still highlyactive in systems where CD4þ T cells are absent (5), suggestingthat the augmentation of recipient DC-induced clonal deletionis highly relevant to the protection seen with this strategy. DCsare specialized cells that are highly efficient at presentinglimiting quantities of antigen and their paradoxical ability todecrease alloreactive T-cell responses reflects the nonphysiolo-gic nature of transplantation. Importantly, the stimulatorycapability of recipient DCs, including their secretion of IL12,is markedly augmented by TBI (12). Furthermore, unlike path-ogen-derived antigen, alloantigen is ubiquitous, present inexcess, and persists indefinitely.

It is clear from our data that both PT-Cy and Pre-T Flt-3L haveimportant implications for GVL effects, and their use for GVHDprevention must be balanced against the inevitable impact onGVL activity. Thus, only well-designed and large prospective

clinical studies can analyze the relative impact of these com-parative GVHD prophylaxis strategies on GVL effects andrelapse after BMT. Given that Flt-3L is a myeloid growth factor,its receptor (Flt3) is ubiquitously expressed by AML blasts (44),and that Flt3 mutations portray an adverse prognosis in AML(45), we would not advocate its use in patients undergoingBMT for myeloid malignancies. Instead, we propose that initialtrials be undertaken in patients with lymphoid malignancies. Inaddition, we hypothesize that Pre-T Flt-3L, like PT-Cy, may bemost appropriate for use in haploidentical BMT, where HLAmismatches are permissive of strong GVL effects mediated bylow numbers of donor T cells escaping deletion. These strate-gies would also seem particularly suited to nonmalignantconditions, indolent hematopoietic malignancies, or settingswhere an engineered graft is employed (e.g., CAR or suicide-gene T cells; ref. 46) such that there is little reliance on donor Tcells within the graft for antitumor effects. Our data demon-strating maintenance of CMV-specific memory T cells with theFlt-3L approach may mean that this strategy results in lowerinfection risk in the clinic, when compared with other T-celldepletion approaches that inevitably target both allo-specificand bystander T cells.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.A. Markey, M.A. Degli-Esposti, G.R. HillDevelopment of methodology: K.A. Markey, S.W. Lane, G.R. HillAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.A. Markey, R.D. Kuns, D.J. Browne, K.H. Gartlan,R.J. Robb, J.P.Martins, S.A.Minnie,M.Cheong,M.Koyama, R.J. Steptoe,G. Belz,M.A. Degli-EspostiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.A. Markey, K.H. Gartlan, A.S. Henden, M. Koyama,G. Belz, M.A. Degli-EspostiWriting, review, and/or revision of the manuscript: K.A. Markey, R.D. Kuns,K.H. Gartlan, R.J. Robb, M.J. Smyth, G. Belz, M.A. Degli-Esposti, S.W. Lane,G.R. HillAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.J. Browne, M.J. SmythStudy supervision: G.R. HillOther (provision of mice): T. Brocker

AcknowledgmentsThe authors would like to acknowledge the assistance of Grace

Chojnowski, Paula Hall, and Michael Rist. T. Brocker is supported by DFGSFB 1054 B03. The project was supported by grants from the NHMRC,Australia.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 25, 2017; revised November 1, 2017; accepted January 8, 2018;published OnlineFirst January 24, 2018.

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2018;24:1604-1616. Published OnlineFirst January 24, 2018.Clin Cancer Res Kate A. Markey, Rachel D. Kuns, Daniel J. Browne, et al. Posttransplant Cyclophosphamide for the Prevention of GVHDAlloreactive Donor T Cells and Represents an Alternative to

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