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Maturation of Plasmacytoid Dendritic CellsGraft-versus-Host Disease Prevents the

MacDonald and Geoffrey R. HillDegli-Esposti, Christian R. Engwerda, Kelli P. A.Olver, Neil C. Raffelt, Alistair L. Don, Mariapia A. Tatjana Banovic, Kate A. Markey, Rachel D. Kuns, Stuart D.

http://www.jimmunol.org/content/182/2/912doi: 10.4049/jimmunol.182.2.912

2009; 182:912-920; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/182/2/912.full#ref-list-1

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Graft-versus-Host Disease Prevents the Maturation ofPlasmacytoid Dendritic Cells1

Tatjana Banovic,* Kate A. Markey,* Rachel D. Kuns,* Stuart D. Olver,* Neil C. Raffelt,*Alistair L. Don,* Mariapia A. Degli-Esposti,† Christian R. Engwerda,* Kelli P. A. MacDonald,2*and Geoffrey R. Hill2,3*

The role of Ag presenting cell subsets in graft-versus-host disease (GVHD) remains unclear. We have thus examined the abilityof plasmacytoid dendritic cells (pDC) to modulate transplant outcome. Surprisingly, host pDC were exquisitely sensitive to totalbody irradiation and were depleted before transplantation, thus allowing us to focus on donor pDC. The depletion of all pDC frombone marrow grafts resulted in an acceleration of GVHD mortality while the depletion of mature pDC from G-CSF mobilizedsplenic grafts had no effect. Thus, donor bone marrow pDC, but not mature pDC contained within stem cell grafts attenuate acuteGVHD. In the presence of GVHD, donor pDC completely failed to reconstitute although a CD11clow120G8� precursor DCreconstituted in an exaggerated and transient manner. These cells expressed Flt-3, the macrophage colony stimulating factorreceptor and, consistent with a common dendritic cell (DC) precursor, were capable of differentiation into pDC and conventionalDC in vivo in the absence of GVHD. These precursors were MHC class II� and CD80/86� but lacked CD40, were activelypresenting host Ag and inhibited GVHD and T cell proliferation in a contact-dependent fashion. These data demonstrate thatGVHD prevents the maturation of pDC and instead promotes the generation of a suppressive precursor DC, further contributingto the state of immune paralysis after transplantation. The Journal of Immunology, 2009, 182: 912–920.

A llogeneic bone marrow transplantation (BMT)4 is the de-finitive curative therapy for the majority of hematolog-ical malignancies and severe immunodeficiencies. A

major complication of allogeneic BMT remains graft-vs-host dis-ease (GVHD) in which the skin, GI tract, and liver are preferen-tially damaged by the transplanted donor immune system. GVHDoccurs in the majority (50–70%) of recipients (1) and is largelyresponsible for the high mortality associated with allogeneic BMT.

The generation of T cell-mediated alloimmune responses aredependent on Ag presentation, cognate interactions between donorT cells and APCs, and the concomitant production of soluble and

membrane-bound costimulatory molecules by APCs and T cells(reviewed in Ref.2)). The initiation of GVHD is absolutely depen-dent on APCs interacting with mature naive donor T cells in thegraft (2). APC include a number of subsets, most broadly dendriticcells (DC), B cells and macrophages. Because DC are the onlysubset capable of inducing responses in naive T cells they havelong been considered the critical cell population in the initiation ofGVHD although unequivocal evidence of this is still lacking. DCcan be discriminated into those that reside in lymphoid organs vstissue and broadly into conventional and plasmacytoid subsets.Conventional DC (cDC) are CD11chigh and MHC class IIhigh and,when mature, initiate potent immune responses. In the mouse, cDCmay be CD4�, CD8�, or negative for both markers. In contrast,plasmacytoid DCs (pDC) are CD11cint, express alternative char-acteristic markers, principally B220, Gr-1 and Ly6C, and producelarge amounts of IFN-� in response to TLR9 signaling. In numer-ous disease models and in vitro culture systems, pDC have beenshown to be capable of inducing Th2 and Tr1 differentiation (3, 4).The aforementioned markers are broadly expressed on B cells,granulocytes, and memory CD8 T cells and so are obviously notexclusive to pDC. Recently, Abs specific for naive pDC have beengenerated that recognize the bone marrow stromal cell Ag-2(PDCA-1 and 120G8 Ab) or Siglec-H (440c) that deplete pDC orblock Siglec-H signaling respectively when administered in vivo(5, 6).

The role of pDC in transplantation and GVHD remains obscure.Certainly the TCRnegCD8� facilitating cell population known tobe permissive of engraftment (i.e., capable of overcoming graftrejection) contains a large proportion of pDC precursors that them-selves appear capable of promoting engraftment (7). However, itremains somewhat controversial as to whether this population or acoexistent CD3� population therein is the dominant facilitatingcell (8, 9). In the setting of GVHD, pDC are known to be mobi-lized by G-CSF, and it has long been suggested that this population

*The Queensland Institute of Medical Research Queensland, Australia; and †TheLions Eye Institute, Nedlands Western Australia, Australia

Received for publication June 27, 2008. Accepted for publication November 5, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 G.R.H. is a National Health and Medical Research Council Practitioner Fellow.K.P.A.M. is a National Health and Medical Research Council R.D. Wright Fellow.This work was supported by grants from the National Health and Medical Re-search Council.

T.B. designed and performed research, analyzed the data and contributed to manu-script preparation; K.A.M. designed and performed research; R.D.K. performed re-search; N.C.R. performed research; S. D. O. performed research; A.L.D. performedresearch; M.A.D.-E. contributed reagents and intellectual input; C.R.E. contributedintellectual input and experimental design; K.P.A.M. contributed intellectual in-put, experimental design, and performed research; and G.R.H. contributed intel-lectual input, experimental design, and contributed to manuscript preparation.2 K.P.M. and G.R.H. contributed equally to this work.3 Address correspondence and reprint requests to Dr. Geoffrey Hill, Bone MarrowTransplantation Laboratory, Queensland Institute of Medical Research, 300 HerstonRoad, Herston, Queensland, Australia. E-mail address: geoffH@qimr.edu.au4 Abbreviations used in this paper: BMT, bone marrow transplantation; GVHD, graft-versus-host disease; DC, dendritic cell; cDC, conventional DC; MLC, mixed lym-phocyte culture; pre-DC, precursor DC; pDC, plasmacytoid DC; SCT, stem cell trans-plantation; TBI, total body irradiation.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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is responsible for the propensity of G-CSF to induce Th2 differ-entiation and attenuate acute GVHD (10). The adoptive transfer ofpurified pDC is laborious and technically challenging in murinesystems and typically requires an in vivo expansion step to gen-erate sufficient cells to harvest from lymphoid organs. Using thisapproach, we have previously demonstrated that the adoptivetransfer of mature pDC from cytokine mobilized animals did notattenuate and in fact exacerbated acute GVHD (11). We have nowtaken advantage of new, more specific reagents to address the roleof pDC in allogeneic stem cell transplantation more methodically.These studies clearly demonstrate the ability of donor precursorbut not mature pDC to suppress GVHD.

Materials and MethodsMice

Female C57BL/6 (B6, H-2b, CD45.2�), B6 Ptprca Ly-5a (H-2b, CD45.1�),B6D2F1 (H-2b/d, CD45.2�), and BALB/c (H-2d, CD45.2�) mice were pur-chased from the Animal Resource Centre. IL-10�/� (B6, H-2b, CD45.2�)mice (12) were bred in the Herston Medical Research Centre (University ofQueensland, Brisbane, Australia). TEa transgenic mice on a B6 back-ground were provided by Jonathan Bromberg (Mount Sinai School of Med-icine, New York, NY). FoxP3.EGFP transgenic mice were supplied byA.Y. Rudensky (University of Washington, Seattle, WA). Mice werehoused in sterilized microisolator cages and received normal chow andacidified water post BMT.

Bone marrow transplantation

Well-established murine transplant models were used and mice were trans-planted according to a standard protocol as previously described (11, 13–15). Recipient B6D2F1 or BALB/c mice received 1100cGy-1300cGy or900cGy respectively (split dose separated by 3 h on day 0) and were trans-planted with either 5 � 106 bone marrow and 2 � 106 purified splenic Tcells or 107 G-CSF mobilized splenocytes from B6 (allogeneic) or B6D2F1(syngeneic) donors. In some experiments non-GVHD controls received Tcell depleted allogeneic bone marrow or G-CSF-mobilized splenocytes.For total T cell depletion, grafts were incubated with hybridoma superna-

tants containing anti-CD4 (RL172), anti-CD8 (TIB211), and Thy1.2 (HO-13-4) mAbs followed by incubation with rabbit complement (CederlaneLaboratories) as previously described (11). Resulting cell suspensions con-tained �1% contamination of viable CD3� T cells. G-CSF (Amgen) wasadministered in some experiments to donors at 10mcg/dose/day subcuta-neously on days �6 to �1. In brief, 120G8 or control Abs (MAC49) wereadministered at 0.5 mg or 1 mg per dose by i.p injection as detailed inresults.

Assessment of GVHD

The degree of systemic GVHD was assessed by a scoring system that sumschanges in five clinical parameters: weight loss, posture (hunching), activ-ity, fur texture, and skin integrity (maximum index � 10) (16). Animalswith severe clinical GVHD (scores �6) were sacrificed according to eth-ical guidelines and the day of death deemed to be the following day, aspreviously described (11, 13, 14, 17).

mAbs and cytokine analysis

The following mAbs were purchased from BioLegend: FITC-conju-gated I-A/I-E (M5/114.15.2), CD45.1 (A20), and IgG2a isotype control;PE-conjugated CD4 (RM4-5), CD8a (53-6.7), NK1.1 (PK136), Sca-1(D7), CD11b (M1/70), CD11c (N418), CD45R/B220 (RA3-6B2),CD45.2 (104), and IgG2b isotype control; PE-Cy5-conjugated CD8,CD4, and IgG2b isotype control; PE-Cy-7-conjugated c-kit (2B8); Al-exa647-conjugated anti-FoxP3 and isotype control; allophycocyanin-conjugated CD11c (N418) and IgG2b isotype control. FITC-conjugatedV�6 (RR4-7), Ly6C (AL-21), CD25 (7D4) and PE-conjugated CD40(3/23), V�2 (B20.1), CD80 (16-10A1), CD86 (GL1), CD62L (MEL14)and I-A/I-E (2G9) were purchased from BD Pharmingen. PE-conju-gated Flt-3 (A2F10) and CD115 (AF598) were purchased from eBio-science. PE-conjugated PDCA-1 was purchased from Miltenyi Biotec(Germany). FITC-conjugated CD11c (N418), and purified mAb againstCD3 (KT3), CD19 (HB305), CD25 (PC61), rat IgG1 isotype control(MAC-49), Gr1 (RB6-8C5), Thy1.2 (HO-13-4), CD62L (MEL14),Ter119, and Fc�R II/III (2.4G2) were produced in house. 120G8 (Ab-Cys) was FITC conjugated in house. Cytokines were analyzed by cy-tokine bead array (BD Pharmingen) as per the manufacturer’sinstructions.

FIGURE 1. pDC before and after TBI or Ab-dependent depletion and effects on acute GVHD. A, Density gradient enriched pDC (gated asCD11cint120G8�) from bone marrow and spleen were phenotyped and MFI is shown in top right. Open histograms are isotype controls. B, Densitygradient enriched cDC (CD11chigh) and pDC (CD11cint120G8�) within host B6D2F1 spleen before and after TBI. C, pDC (CD11cint120G8�) withinhost bone marrow 24 h after 120G8 or control Ab administration. Mean � SE of percentage of pDC shown (n � 7 per group). D, Survival by KaplanMeier analysis following transplantation of pDC replete or depleted bone marrow and purified splenic T cells (2 � 106/animal) from B6 donors intolethally irradiated (1300 cGy) B6D2F1 recipients (n � 14 per group). Donors received 120G8 or control Ab 24 h earlier and non-GVHD controlrecipients were transplanted with syngeneic B6D2F1 grafts (n � 8). Data combined from two replicate experiments, ��, p � 0.012, B6 BM � T,120G8 vs control Ab.

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Cell preparation, culture, and depletion

DC purification was undertaken as previously described (18). In brief, low-density cells were enriched from digested spleen by Optiprep density gra-dient centrifugation. For culture experiments, highly purified DC popula-tions (�98%) were obtained from the density-gradient enriched APCfraction by fluorescence-activated cell sorting (MoFlo, DakoCytomation)of precursor DC (pre-DC) (CD11clow/PDCA-1� or CD11clow/120G8�

cells). Splenic CD3� T cells were purified to �90% by depleting B cells(B220, CD19), monocytes (CD11b), granulocytes (Gr-1), and erythroidcells (Ter119) using magnetic bead depletion. Naive T cells were purifiedfrom spleen for in vitro assays by sorting of the Thy1.2�CD62L� popu-lation. Transgenic TEa cells were sort purified on the basis of V�2 andV�6 to �99% purity. In in vitro culture experiments using pre-DC, 106

sort-purified pre-DC (CD11clow/120G8�) were cultured in 24-well platessupplemented with murine Flt-3L (300 ng/ml) and IL-3 (50 ng/ml) for 8–9days as previously described (19, 20). CFSE (Molecular Probes) labelingof T cells was performed as described (21). In brief, T cells were resus-pended at a density of 3 � 107 cells/ml in RPMI 1640 and CFSE was addedat final concentration of 2 �M. Cells were analyzed using FACSCalibur(BD Biosciences) and the data were processed with ModFit LT cell cycleanalysis software (Verity Software House). For mixed lymphocyte culture(MLC) studies, naive (CD62Lhighthy1�) B6 T cells were labeled withCFSE and T cells were plated with B6D2F1 sort-purified (CD11chigh/classIIhigh) cDC with or without sort purified donor (B6) pre-DC from animalswith GVHD in the presence of IL-3 (12.5 ng/ml). Transwell plates (Corn-

ing) were used to establish the contact-dependence of pre-DC inducedsuppression and proliferation was assessed after 4 or 5 days of culture. CpGstimulation and latex bead uptake was undertaken as previously described(13).

Phenotypic analysis

For analysis of cell surface molecules, cells were incubated with mAb asper the manufacturer’s instructions, or at 5–20 �g/ml in 2% FCS in PBS,for 20–60 min at 4°C. Cells were washed in 2% FCS in PBS. BiotinylatedmAb were detected with the appropriate conjugated streptavidin detectionreagents diluted in 2% FCS in PBS and analyzed on a FACSCalibur (Bec-ton Dickinson). 7-AAD (1 �g/ml) was added in the final wash to identifydead cells.

Microscopy

Images of cytospin cell samples were acquired on an Olympus BX41 mi-croscope and Olympus DP12 camera (Japan) using a �10 ocular objectiveand �100/1.25 oil objective at room temperature.

Statistics

Survival curves were plotted using Kaplan-Meier estimates and comparedby log-rank analysis. The Mann-Whitney-U test was used for the statisticalanalysis of all other data. p � 0.05 was considered statistically significant.

FIGURE 2. pDC depletion of G-CSF mobilized spleen and effects on acute GVHD. A, Density gradient enriched splenic pDC (CD11cint120G8�) fromB6 donors treated with G-CSF (10 mcg/day) on days �6 to �1. 120G8 Ab was administered on day �7 and mean pDC depletion documented thereafteras a percentage of absolute number in recipients of control Ab (n � 4–5 per time point). B, Numbers of specific cell lineages per spleen on day 0 in G-CSFmobilized donors that received 120G8 or control Ab at day �7, �4, and �1 (n � 4 to 10 per group). �, p � 0.028, all others are NS. C, Survival by KaplanMeier analysis and GVHD clinical scores following transplantation of pDC replete or depleted G-CSF mobilized splenocytes from B6 donors into lethallyirradiated (1100 cGy) B6D2F1 recipients. Donors received 120G8 or control Ab at various times during G-CSF mobilization and non-GVHD controlrecipients were transplanted with T cell depleted, G-CSF mobilized splenocytes (n are as shown). Data combined from six experiments, no significantdifferences in survival curves.

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ResultspDC within bone marrow grafts attenuate GVHD

We first studied the profile of bone marrow vs splenic pDC. Asshown in Fig. 1A, bone marrow pDC had lower expression ofCD40, CD80, CD86, and MHC class II than splenic pDC. Whenwe analyzed splenic (and marrow, data not shown) DC before and24 h after TBI, we were surprised that pDC were already com-pletely depleted (Fig. 1B). In contrast, cDC still remained, albeit inreduced numbers, as previously described (15, 22). Because recip-ient pDC were unlikely to influence the initiation of GVHD fol-lowing myeloablative TBI doses, we focused on the ability ofdonor derived pDC to influence alloreactivity. We used the pDC-specific Ab 120G8 to deplete pDC and study the effect of this cellpopulation within bone marrow grafts on subsequent GVHD. Asshown in Fig. 1C, administration of this Ab to donors depletedbone marrow pDC by �80%. When these depleted B6 grafts weretransplanted into lethally irradiated B6D2F1 recipients they in-duced significantly greater levels of GVHD than grafts from non-depleted, control Ab-treated donors (Fig. 1D). Because stem cellmobilization with G-CSF has been shown to increase pDC num-bers in the peripheral blood and this has been postulated as one ofthe regulatory mechanisms invoked by this cytokine, we next useda stem cell transplant model. In this system, pDC were depletedthroughout or at the end of mobilization and splenic grafts subse-quently transplanted using the same B63 B6D2F1 strain combi-nation. Donor pDC were depleted throughout the mobilization

since they began to reconstitute within 48 h of 120G8 admin-istration (Fig. 2A). The depletion process did not deplete othercell populations within the graft (Fig. 2B). Surprisingly the de-pletion of mature pDC from mobilized splenic grafts had noeffect on GVHD mortality or clinical scores (Fig. 2C). Thesedata confirm that bone marrow pDC are indeed immunomodu-latory and suppress GVHD while this property is lost as thesecells mature in the periphery.

Reconstitution of donor DC after bone marrow transplantation

We next studied the kinetics of donor pDC reconstitution afterBMT. Surprisingly there was an almost complete failure of pDCmaturation in animals with GVHD, as opposed to the normalreconstitution in non-GVHD recipients of T cell depleted grafts(Fig. 3, A–C). In contrast, cDC reconstituted in a relativelynormal fashion (Fig. 3, A–C) although at later time points theirnumbers were reduced in conjunction with the lymphoid atro-phy seen in animals with GVHD. Interestingly, in animals withGVHD, a CD11clow/PDCA-1� or CD11clow/120G8� cell withplasmacytoid morphology (Fig. 3, B and C) reconstitutedquickly after BMT and stem cell transplantation, peaking at day7–14 (Fig. 3, B and C). Thereafter, they diminished rapidly andbeyond day 28, when GVHD mortality was maximal, they werelargely absent. These cells were clearly distinguishable frommature pDC seen in syngeneic animals (which had a classical

FIGURE 3. Donor pDC reconstitution after transplantation. A, Density gradient enriched cDC (CD11chigh120G8�) and pDC (CD11cint120G82� andCD11cintB220high) in animals with GVHD and non-GVHD controls receiving T cell replete or depleted G-CSF mobilized splenocytes. Note that in animalswith GVHD there is a failure of pDC maturation and the presence of a CD11clow120G8� population (pre-DC) early after transplant. Identical profiles wereseen in recipients of bone marrow and T cell grafts. B, Numbers of reconstituting donor pre-DC, pDC, and cDC cells in the spleen of animals with andwithout GVHD (n � 3–6 per group) over time in recipients of bone marrow transplants with or without splenic T cells as shown. ND refers to the factthat mature pDC are not detectable or below scale in animals with GVHD. One of three experiments is shown and is representative of data generated inboth stem cell and bone marrow transplant models. C, Morphology (�400) of pre-DC from animals with GVHD 7 days after BMT. Morphology of pDCand cDC is from non-GVHD animals seven days after BMT.

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pDC phenotype (23)) on the basis of morphology and their re-duced expression of CD11c, 120G8, and, most importantly, theabsence of B220 expression). These cells reconstituting duringGVHD are herein referred to as pre-DC.

Phenotypic and functional analysis of reconstitutingdonor pre-DC

Because 120G8 can be promiscuously expressed during immuneresponses (5), we more extensively phenotyped the reconstituting“pre-DC” in animals with GVHD. These cells had the character-istic CD11clow, MHC class IIhigh, 120G8�, PDCA-1�, and Ly6C�

phenotype of pDC but lacked B220 (Fig. 4A) and produced largeamounts of cytokines in response to CpG (Fig. 4B). They werehighly phagocytic (Fig. 4C), as has been recently described forpDC (4), and only weakly stimulated responses in allogeneic Tcells (Fig. 4D). Importantly, they were actively presenting host Agbecause they stimulated TEa transgenic T cells (which respondonly to a processed I-Ed peptide presented in I-Ab; Refs. 4, 24 andFig. 4E). Notably, the cells did not produce IFN-� in response toCpG motifs (data not shown). Although they expressed interme-diate levels of Flt-3 and M-CSFR, consistent with common DCprecursors (25), a proportion also expressed CD11b suggestingthey were, at least partially, differentiating down a monocytic lin-eage. However, after culture in IL-3 and Flt-3L, the majority of

these precursor cells differentiated into CD11c�PDCA-1� cells ofclear plasmacytoid morphology that were Siglec-H� (Fig. 5A), allcharacteristic of mature pDC. A smaller fraction matured intogranulocytes that were Siglec-Hneg, suggesting heterogeneity inthe precursors contained within the initial population. To study thematuration of these cells in vivo in the absence of GVHD, con-genic CD11cint/120G8� cells reconstituting during GVHD weresort purified 7 days after BMT and added to syngeneic bone mar-row grafts so that maturation could be followed in vivo. The ma-jority (70%) of these cells differentiated into DC with both cDCand pDC represented (Fig. 5B), confirming the original CD11clow/120G8� population was indeed highly enriched for a common DCprecursor.

Reconstituting DC precursors are potent suppressors ofallogeneic T cell responses

We next sought to investigate the suppressive properties of recon-stituting DC precursors (pre-DC) by adding them to MLC. TheMLC was used as a surrogate to study the ability of pre-DC tosuppress allogeneic T cell responses to host Ag. After BMT, thisAg presentation may be occurring by host APC (which can persistlong term in BMT recipients within tissue; Ref. 26) or by donorAPC that present exogenous host Ag, primarily within MHC classII (27). As shown in Fig. 6, A and B, reconstituting donor DC

FIGURE 4. Phenotype and function of reconstituting donor pre-DC. A, Phenotype of 120G8� pre-DC in animals with GVHD. B, Cytokine generationfrom sort purified 120G8� pre-DC from animals with GVHD 7 days after BMT in response to 18 h stimulation with CpG. C, Intense phagocytosis of latexbeads in overnight cultures by reconstituting donor pre-DC (�1000). D, Relative capacity of B6 (H-2b) naive pDC and the pre-DC reconstituting in animalswith GVHD to stimulate alloreactive proliferation in BALB/c (H-2d) T cells in IL-3 supplemented MLC. Proliferation was determined by [3H]thymidineincorporation and results expressed as mean � SE of triplicate wells. One of two replicate experiments. E, B6 bone marrow and T cells were transplantedinto lethally irradiated BALB/c recipients. Reconstituting donor pre-DC were sort purified and used to stimulate TEa transgenic T cells (that recognizeprocessed I-Ed Ag in the context of I-Ab). B6D2F1 splenocytes were used as a positive control and BALB/c and B6 splenocytes as negative controls.Proliferation determined by [3H]thymidine incorporation and results expressed as mean � SE of triplicate wells. One of two replicate experiments shown.

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precursors dramatically suppressed T cell responses to allogeneicDC, at ratios of pre-DC to T cells from 1:1 to at least 1:16 (data notshown). This inhibition was independent of IL-10, TGF�, IFN-�,or IDO as it was present when the pre-DC were derived fromIL-10�/� donors or when TGF�, IFN-�, or IDO were inhibited byAb or 1-methyl tryptophan (data not shown). Consistent with this,the inhibition could not be transferred by addition of supernatantsfrom a “suppressed” MLC where pre-DC were present, to a secondMLC containing only T cells and allogeneic DC (Fig. 6C). Thissuppression did not require FoxP3� regulatory T cells since sup-pression by pre-DC remained when they were removed from the Tcell inoculum by sort exclusion of EGFP� cells derived fromB6.FoxP3-EGFP transgenic T cells (Fig. 6D). Furthermore, the useof split well MLCs confirmed that the DC precursors required cellcontact to suppress allogeneic T cell responses (Fig. 6E). Finally,when reconstituting DC precursors were depleted in vivo by ad-ministration of 120G8 after BMT, there was a marked accelerationof GVHD from day 28 forward to day 7–14 after BMT (Fig. 7A).Thus, the depletion of pre-DC reconstituting from bone marrowduring GVHD further significantly accelerated GVHD over thatseen when only bone marrow pDC were depleted, confirming thatpre-DC themselves exert a suppressive effect in vivo. Importantly,

the depletion process was specific and did not influence the recon-stitution of other donor cell populations in vivo (Fig. 7B), con-firming that these donor DC precursors were indeed suppressive invivo. Additionally, donor T cells from animals in which pre-DCwere depleted demonstrated enhanced proliferative and Th1 re-sponses to host Ags (Fig. 7C).

DiscussionThe importance of the contribution of APC to the induction ofacute GVHD has become clearer over the last decade. It is nowestablished that host APC are critical for the induction of CD8dependent acute GVHD while either host or donor are sufficient toinduce CD4-dependent disease (28, 29). The subsets of APC im-portant in this process remain less clear although increasing cir-cumstantial evidence suggests that mature conventional DC are atleast one important stimulatory population (22, 30). Conversely, Bcells seem to have a limited capacity to induce acute GVHD (asopposed to chronic GVHD) and instead appear to possess suppres-sive properties (31). The increase in circulating donor pDC duringstem cell mobilization with G-CSF has been associated with Th2differentiation and a reduction in the potential of donor T cells toinduce acute GVHD on a per cell basis (10) although this link has

FIGURE 5. Differentiation of the reconstituting donor pre-DC. A, Sort-purified 120G8� pre-DC from animals with GVHD 7 days after BMT werecultured in murine Flt-3L (200 ng/ml) for 8 days in vitro. Cells were phenotyped at the end of culture for CD11c and Siglec-H and the two fractionsexamined morphologically (�400). One of three experiments shown. B, 106 sort purified donor B6 CD45.1�120G8� pre-DC from animals with GVHD7 days after BMT were added to 106 B6 CD45.2� bone marrow and transplanted into lethally irradiated B6 CD45.2� recipients. Nine days later, thedifferentiated density enriched CD45.1� cells were phenotyped for pDC markers. One of two experiments shown.

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not been causally established in vivo. The data provided hereformally establish this link and further extend this paradigm, con-firming that it is bone marrow derived DC precursors that are im-portant for attenuation of GVHD.

The ability of pDC to modulate immunity has been widely de-scribed and in the transplant setting, this effect has been predom-inantly suppressive in nature, usually via the induction of classicalor induced regulatory T cells (3, 4). Furthermore, this pathway hasrecently been described as IDO dependent (32) and the pDC pro-ducing high levels of IDO are a CD19� subset which in turn in-duce high levels of PD-L1/PD-L2 expression on conventional DC.We were unable to demonstrate a significant population of CD19�

mature pDC from the spleen or lymph nodes of our donor animalsand this did not appear to be a suppressive pathway in our trans-plant system. Conversely, the suppressive bone marrow-derivedDC precursors did not express CD19 by flow cytometry or IDO byreal time PCR, nor did they themselves express or induce PD-L1/PD-L2 on conventional DC (data not shown). Thus, it appearsunlikely that IDO or PD-L1/PD-L2 are the major suppressive path-ways invoked by these cells. Likewise, TGF�, IL-10, or IFN-�appear redundant for this suppressive activity. Importantly, the do-nor DC precursors were highly phagocytic and were actively ex-pressing processed host alloantigens, albeit in the absence ofCD40. The induction of tolerance by “regulatory DC” that expressAg in the absence of CD40 in vivo is widely described (13, 33, 34)and, at present, this appears the dominant pathway of suppression

(i.e., anergy) invoked by these cells. It should be noted howeverthat the requirement for contact-dependent suppression is not pos-sible to validate in vivo. The finding that these immature DC pre-cursors are suppressive in nature is consistent with the describedability of immature DC to inhibit T cell responses (33, 35, 36).

The lineage of pDC has been debated considerably in the recentpast and it is clear that both common myeloid and lymphoid pro-genitors can generate pDC. However, at steady state, pDCs appearto be predominantly myeloid in origin (37). Furthermore, a com-mon precursor, expressing both the M-CSF receptor (c-fms) andFlt-3 has recently been described that is capable of generating bothcDC and pDC (25, 38). A second macrophage, DC precursor withsimilar phenotype, has also been described (39) that likely repre-sents an overlapping population (40). The bone marrow-derivedsuppressive precursor population identified in these studies in-cludes large numbers of this population, as demonstrated by phe-notypic analysis and the demonstration of preferential differenti-ation to both DC subsets in irradiated syngeneic recipients. This isconsistent with the broad definition of a precursor DC as describedby Shortman (41). However, it should be noted that the majorityof the DC precursors defined to date are within steady state con-ditions, not the profoundly inflammatory setting of GVHD encoun-tered in these studies. We and others (13, 42) have demonstratedthe ability of suppressive myeloid granulocyte-monocyte precur-sors within the donor graft to attenuate GVHD and the IL-10 de-pendence of this process. Importantly, the pre-DC described in this

FIGURE 6. Suppression of alloreactive T cell responses by reconstituting donor pre-DC. A, 105 B6 (H-2b) CFSE-labeled CD45.1�Thy1�CD62L� naiveT cells were cultured in MLCs with 104 B6D2F1 (H-2d) cDC, with or without 104 sort purified pre-DC from animals with GVHD 7 days after BMT. CFSEdilution in CD4 and CD8 T cells was determined 96 h later. One of six experiments shown. B, Proliferation indices from A as determined by Modfit analysis.Data are mean � SE from triplicate wells. C, MLC were undertaken with cDC and T cells as in A with or without the addition of tissue culture supernatants(TCSN) from a primary MLC containing pre-DC. One of two replicate experiments shown. D, Pre-DC were added to MLC in which T cells contained botheffector T cells and FoxP3� regulatory T cells or effector T cells only (by sort exclusion of EGFP� cells derived from B6.FoxP3-EGFP transgenic T cells).Similar data was also obtained by (FACS) removal of CD4�CD25high T cells. E, MLC were undertaken as in A, with pre-DC present in the upper chamber(UC) or lower chamber (LC) of transwell plates. One of two replicate experiments shown.

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study are not conventional myeloid suppressor cells based on size,morphology, their MHC class IIhigh/CD11c�/120G8� phenotype,the fact that they are CD31low (data not shown) and their ability togenerate pDC in vivo. In addition, this phenotype and function isnot characteristic of inflammatory monocytes (41, 43) and insteadis most consisted with a pre-DC corrupted in maturation by theGVHD milieu.

To our knowledge this is the first description of a specific failureof pDC maturation during GVHD (as opposed to the global lym-phoid atrophy more characteristic of GVHD). This suggests thatGVHD either provides potent inhibitory signals to maturation, oralternatively, generates a cytokine milieu that is lacking a criticalpDC growth factor. The best described growth factor for both DCsubsets is Flt-3L and to a lesser extent, GM-CSF and M-CSF (25,44). Certainly the provision of Flt-3L in vitro (and removal fromthe GVHD environment) did allow the generation of Siglec-H�

plasmacytoid cells while in vivo full cDC and pDC maturationoccurred. Thus, it appears that the additional signals required toallow the full differentiation of DC precursors are absent or per-haps more likely, additional inflammatory stimuli corrupt normalcDC and pDC differentiation.

The promotion of tolerance remains the ultimate therapeuticgoal in transplantation. These studies highlight and delineate mar-row derived pDC and pre-DC as important cell populations in thesuppression of this process. Because these cells rely on Flt-3L andM-CSF as growth factors (44), this raises important questions inrelation to the use of these growth factors to expand this populationwithin the marrow before transplantation in contrast to the simplemobilization into the periphery with G-CSF. Consistent with this,the administration of Flt-3L to BMT recipients has already been

demonstrated to suppress the development of acute GVHD (45).Importantly, the impairment of cDC and pDC maturation byGVHD may be responsible for the severe immune paralysis asso-ciated with this disease and delineation of the pathways involvedmay allow therapeutic intervention to circumvent the severe infec-tive mortality and morbidity associated with GVHD.

AcknowledgmentsWe thank Professor Ken Shortman (Walter and Elisa Hall Institute, Mel-bourne, Victoria, Australia) for his helpful discussions regarding the DCidentified within this work.

DisclosuresThe authors have no financial conflict of interest.

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