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TRANSPLANTATION
Organ-derived dendritic cells have differential effects on alloreactive T cells
Theo D. Kim,1 Theis H. Terwey,1 Johannes L. Zakrzewski,1 David Suh,1 Adam A. Kochman,1 Megan E. Chen,1
Chris G. King,1 Chiara Borsotti,1 Jeremy Grubin,1 Odette M. Smith,1 Glenn Heller,2 Chen Liu,3
George F. Murphy,4 Onder Alpdogan,1 and Marcel R. M. van den Brink1
1Department
of Medicine and Immunology, and 2Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY;
of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville; and 4Department of Pathology,
Brigham and Women’s Hospital, Boston, MA
3Department
Dendritic cells (DCs) are considered critical for the induction of graft-versus-host
disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been
shown to induce organ-tropism through
induction of specific homing molecules
on T cells. Using adoptive transfer of
CFSE-labeled cells, we first demonstrated
that alloreactive T cells differentially upregulate specific homing molecules in
vivo. Host-type dendritic cells from the
GVHD target organs liver and spleen or
skin- and gut-draining lymph nodes effectively primed naive allogeneic T cells in
vitro with the exception of liver-derived
dendritic cells, which showed less stimulatory capacity. Gut-derived dendritic cells
induced alloreactive donor T cells with a
gut-homing phenotype that caused increased GVHD mortality and morbidity
compared with T cells stimulated with
dendritic cells from spleen, liver, and peripheral lymph nodes in an MHCmismatched murine BMT model. However, in vivo analysis demonstrated that
the in vitro imprinting of homing molecules on alloreactive T cells was only
transient. In conclusion, organ-derived
dendritic cells can efficiently induce specific homing molecules on alloreactive T
cells. A gut-homing phenotype correlates
with increased GVHD mortality and morbidity after murine BMT, underlining the
importance of the gut in the pathophysiology of GVHD. (Blood. 2008;111:2929-2940)
© 2008 by The American Society of Hematology
Introduction
Allogeneic hematopoietic stem-cell transplantation is an important
therapeutic option for a variety of malignant and nonmalignant
diseases.1 However, the occurrence of acute graft-versus-host
disease (GVHD), a serious systemic illness primarily involving the
intestine, liver, and skin, constitutes one of the limitations for its
widespread use.2,3 GVHD occurs when alloreactive donor T cells
recognize alloantigens on normal host tissues. Pathophysiologically, 3 distinct phases (as defined by Ferrara and Reddy) can be
distinguished, which are characterized by tissue injury from the
conditioning regimen, donor T-cell activation, and efferent effector
functions.4 The gastrointestinal tract hereby seems to play a crucial
role in the amplification of systemic disease by permitting release
of inflammatory stimuli from the gut.5
To mount an efficient alloresponse, antigens need to be presented by professional antigen presenting cells (APCs) with host
APCs being crucial for the initiation of GVHD.6-10 Depletion of
APCs in the liver and the spleen significantly inhibits the recruitment and proliferation of donor T cells,11 and the elimination of
Langerhans cells seems to be efficient in preventing skin GVHD.12
However, there is also increasing evidence implicating crosspresenting donor APCs in acute13 and chronic14 GVHD. The
importance of antigen expression by epithelial, nonhematopoietic
GVHD target organ cells is still being disputed.15,16
Recent studies have shown that dendritic cells not only are
required for the activation of T cells but also influence their
migratory behavior.17-19 Dendritic cells from mesenteric lymph
nodes (MLNs),20,21 Peyer patches,22 and peripheral lymph nodes
(PLNs)23 have all been shown to imprint tissue-specific homing
molecules on T cells. However, this process is flexible and allows
reprogramming after exposure to different dendritic cells.23,24
Whereas gut homing is induced by retinoic acid,25 skin homing
seems to be dependent on vitamin D3.26 More recently, dendritic
cells have also been shown to affect B-cell homing.27
This process of imprinting and the tight control of T-cell
migration is of particular relevance for a number of physiologic and
pathologic conditions.28 Since alloreactive T cells can exert their
pathologic effect only after infiltration into GVHD target organs,
interfering with this process is an attractive approach in preventing
or treating GVHD. The feasibility of this method has been
ascertained in several studies with various molecules.29,30
In this study, we examined the effect of different organderived host-type dendritic cells on alloreactive T cells and
found that dendritic cells derived from the liver had less
allostimulatory capacity than any other organ-derived dendritic
cell. However, dendritic cells from the gut-associated lymphoid
tissue specifically induced a gut-homing phenotype resulting in
significantly higher mortality and morbidity after bone marrow
transplantation. In addition, microarray analysis performed on
those stimulated T cells revealed novel differentially induced
target genes.
Submitted June 19, 2007; accepted December 27, 2007. Prepublished online as
Blood First Edition paper, January 4, 2008; DOI 10.1182/blood-2007-06-096602.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2008 by The American Society of Hematology
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
2929
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2930
KIM et al
Methods
Antibodies and flow cytometry
Most fluorochrome- or biotin-labeled antimurine antibodies were obtained
from BD Biosciences Pharmingen (San Diego, CA) with the exception of
rat antimouse CD16/CD32 (2.4G2), produced by the Monoclonal Antibody
Core Facility at the Memorial Sloan-Kettering Cancer Center. Foxp3
(FJK-16s) and integrin ␣4 chain (R1-2) antibodies were purchased from
eBioscience (San Diego, CA) and antimurine integrin ␤1 chain (Hm␤1-1)
was from BioLegend (San Diego, CA). Staining for E- and P-selectin
ligands was performed using chimeric proteins consisting of the extracellular domains of E- and P-selectin fused to the human Fc region (from BD
Biosciences Pharmingen and R&D Systems, Minneapolis, MN, respectively), which was detected with a secondary phycoerythrin-conjugated
goat antihuman FcR antibody (Jackson ImmunoResearch, West Grove, PA).
Fluorescence-activated cell sorting (FACS) staining was performed as
previously described.31 Samples were acquired on a LSR I cytometer
(Becton Dickinson, San Jose, CA) with CellQuest software (Becton
Dickinson) and analyzed with FlowJo (Tree Star, Ashland, OR).
Analysis of T-cell proliferation in vivo
T cells were positively selected from red blood cell (RBC)–lysed splenocytes using CD5 MicroBeads (Miltenyi Biotec, Auburn, CA) and subsequently stained with 5 ␮M carboxyfluorescein diacetate succinimidyl ester
(CFSE; Molecular Probes, Eugene, OR) for 20 minutes at 37°C. Between
15 and 30 ⫻ 106 stained cells were transplanted into lethally irradiated
allogeneic recipients. Organs were harvested after 72 hours and
analyzed with FACS.
Isolation of dendritic cells
To expand dendritic cells, C57BL/6 mice were injected subcutaneously
with 10 ⫻ 106 B16 melanoma cells transduced with murine FLT3L (kindly
provided by U. von Andrian, the CBR Institute for Biomedical Research,
Harvard Medical School, Boston, MA). After 12 to 17 days, mice were
killed and spleen, PLNs (axillary and inguinal), and MLNs were harvested,
homogenized between frosted slides, and incubated for 15 minutes at 37°C
in digest medium containing 1 mg/mL collagenase D and 100 ␮g/mL Dnase
I (both from Roche, Indianapolis, IN). Livers were digested in Liver Digest
Medium (Gibco, Carlsbad, CA) and nonparenchymal cells isolated by
percoll separation (Amersham Biosciences, Piscataway, NJ). In some cases,
recombinant human FLT3L (generously provided by Amgen, Thousand
Oaks, CA) was given subcutaneously for 10 consecutive days before
harvest of organs. Dendritic cells were purified immunomagnetically by
positive selection with CD11c (N418) MicroBeads (Miltenyi Biotec)
according to the manufacturer’s instructions. Purity was analyzed by FACS
and was between 90% and 95%.
T-cell stimulation
Donor-type T cells (B10.BR) were positively selected using CD5
MicroBeads (Miltenyi Biotec) and cocultured with host-type dendritic
cells for 4 days at a ratio of 2:1 in T-cell medium consisting of RPMI
1640 (Mediatech, Herndon, VA), 10% heat-inactivated fetal bovine
serum (FBS), 0.01 M HEPES, 2 mM L-glutamine, gentamicin (20 ␮g/mL),
and ␤-mercaptoethanol. To increase maturation of dendritic cells, E coli
O111:B4 lipopolysaccharide (LPS; Calbiochem, San Diego, CA) was
added at 100 ng/mL at the start of the culture. In some cases, LPS was
substituted with recombinant murine sCD40L from PeproTech at 1 ␮g/mL
(Rocky Hill, NJ).
Cell proliferation and suppression assay
Various doses of organ-derived allogeneic dendritic cells were cocultured
with 105 splenic donor-type T cells (at ratios of 1:100, 1:10, and 1:1) in
triplicate wells of 96-well plates for 4 days in the presence of 100 ng/mL
LPS. Tritiated thymidine ([3H]thymidine, 1 ␮Ci [0.037 MBq]/well) was
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
added for the last 18 hours of stimulation, and counts per minute were
measured with a Topcount NXT microplate scintillation counter (Packard,
Meridan, CT). For the suppression assay, 105 C57BL/6-derived T cells
(responder) were cultured for 4 days with 2 ⫻ 105 irradiated (2000 cGy)
BALB/c-derived splenocytes as stimulators and 6 ⫻ 105 B10.BR-derived
CD4⫹CD25⫹ regulatory T cells that were isolated using magnetic beads
after 4 days of T-cell stimulation with dendritic cells as described in the
previous paragraph.
Enzyme-linked immunosorbent assay
Concentration of cytokines in cell culture supernatants or sera of mice that
underwent transplantation was determined with commercially available enzymelinked immunosorbent assay (ELISA) kits (R&D Systems) according to the
manufacturer’s protocol. Serum was obtained by retro-orbital bleeding and stored
at ⫺80°C until analysis.
Intracellular cytokine staining
Intracellular cytokine production of in vitro–activated donor T cells was
measured by staining with (surface) fluorochrome-conjugated antibodies, fixing, and permeabilizing with the Cytofix/Cytoperm Kit (Pharmingen). Cells were subsequently stained with intracellular cytokine (IFN-␥
and TNF-␣) PE-conjugated antibodies. In some experiments, T cells
were stimulated with PMA (10 ng/mL) and ionomycin (2 ␮M) for the
final 4 hours, and brefeldin A (10 ␮g/mL) was added for the last 3 hours
of incubation.
RNA isolation, labeling, microarray hybridization,
and data analysis
RNA was extracted from fresh cultures by homogenization in TRIzol
reagent (Gibco). Total RNA (2 ␮g) was used for cDNA synthesis using an
oligo(dT)-T7 primer and the SuperScript Double-Stranded cDNA Synthesis
Kit (Invitrogen Life Technologies, Carlsbad, CA). Synthesis, linear amplification, and labeling of cRNA were accomplished by in vitro transcription
using the MessageAmp aRNA Kit (Ambion, Austin, TX) and biotinylated
nucleotides (Enzo Diagnostics, Farmingdale, NY). Labeled and fragmented
cRNA (10 ␮g) was then hybridized onto a GeneChip Mouse Genome 430A
2.0 Array (Affymetrix, Santa Clara, CA) representing approximately
14 000 mouse genes and scanned with a high-numeric aperture and flying
objective (FOL) lens in a GS3000 scanner (Affymetrix). The image was
quantified using Microarray Suite 5.1 (Affymetrix). Three independent
RNA samples were analyzed for each of the 4 culture conditions. Using
GCOS 1.3 (Affymetrix), we performed a cross comparison of each
experimental sample to each reference. Cutoff values for fold change and
P values were plus or minus 1.25 and .001, respectively. All array data have
been deposited in GEO32 (accession number GSE5503).
Mice and bone marrow transplantation
The bone marrow transplantation (BMT) procedure was performed as
described previously.31 Female C57BL/6 (B6) (H-2b) and B10.BR
(H-2k) mice were obtained from The Jackson Laboratory (Bar Harbor,
ME). All bone marrow transplantation protocols were approved by the
Memorial Sloan-Kettering Cancer Center Institutional Animal Care and
Use Committee.
Assessment of GvHD
Survival was monitored daily, and mice were individually scored weekly
for 5 clinical parameters (weight loss, hunched posture, activity level, fur
ruffling, and skin lesions) on a scale from 0 to 2, as previously described.33
A clinical GVHD score was generated by the summation of the 5 criteria,
and mice scoring 5 or greater were killed.
Histopathologic analysis of target organ GVHD
Mice were killed after BMT for histopathologic analysis of GVHD target
organs (small and large bowel, liver, and skin). Organs were harvested,
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
formalin-preserved, paraffin-embedded, sectioned, and hematoxylin/eosin
stained. Cutaneous GVHD was assessed by determining the number of
apoptotic cells per millimeter of epidermis (G.F.M).34 A semiquantitative
score consisting of 19 to 22 different parameters associated with GVHD
was calculated for liver and gut samples (C.L.).35 Immunohistochemical
staining for CD3 was performed using standard protocols.
DENDRITIC CELLS IMPRINT ALLOGENEIC T CELLS
A
50
Spleen
Liver
PLN
MLN
40
LPAM-1
30
2931
*
*
20
10
0
Assessment of organ infiltration
0
1
2
3
4
5
Cell cycles
For assessment of organ infiltration with donor T cells, recipient mice were killed
after BMT and their target organs (skin, spleen, liver, PLNs, and MLNs)
harvested. Mononuclear cells were isolated using standard procedures.
**
30
20
E-lig
Statistics
10
All values shown in graphs represent the mean plus or minus SEM.
Groupwise comparisons except for survival and GVHD score analysis were
done with the one-way ANOVA test. The Mann-Whitney test or the
Wilcoxon matched pairs test was used where appropriate. Survival data
were analyzed with the Mantel-Cox log-rank test. For GVHD scores and the
change in weight from baseline, the statistical analysis performed to test
whether a differential change occurred between treatment groups was the
difference in the area under curve (AUC) between groups, using all possible
pairwise contrasts. Every mouse could not be followed for the full length of
the study. To account for informative dropouts, the AUCs were calculated
up to the minimum follow-up time for each pairwise difference. A P less
than .05 was considered statistically significant.
0
0
1
2
3
4
5
Cell cycles
250
***
200
150
P-lig
100
50
0
0
1
2
3
4
5
Cell cycles
Homing molecules are differentially up-regulated on allogeneic
donor T cells in vivo
The induction of particular homing molecules on T cells seems to
occur rapidly in secondary lymphoid organs in vivo.36 To determine
whether alloreactive T cells display differential expression of
homing molecules depending on their site of activation, we
analyzed the expression of the gut-homing molecule LPAM-1
(␣4␤7 integrin) and the skin-homing molecules E- and P-selectin
ligands (E-lig and P-lig), primarily the carbohydrate epitope
cutaneous lymphocyte antigen (CLA) and P-selectin glycoprotein
ligand 1 (PSGL1), respectively, in the spleen, liver, PLNs, and
MLNs. Three days after adoptive transfer of CFSE-labeled naive
allogeneic T cells (B10.BR) into lethally irradiated hosts (C57BL/
6), we found significant up-regulation of LPAM-1 on rapidly
dividing alloreactive T cells in MLNs compared with PLNs (Figure
1A,B).37 The expression of E-lig and P-lig was up-regulated in
PLNs but not in MLNs. We conclude that alloreactive T cells in
MLNs up-regulate LPAM-1, whereas alloreactive T cells in PLNs
express more E-lig and P-lig.
Expanded organ-derived dendritic cells are heterogeneous and
phenotypically immature but rapidly mature in the presence of
LPS in vitro
We isolated dendritic cells to better define their contribution to this
induction of homing molecules.17 Administration of FMS-like
tyrosine kinase 3 ligand (FLT3L) was necessary to generate
sufficient numbers of host-type (C57BL/6) dendritic cells for use in
bulk cultures and was given either through implantation of a
murine FLT3L-transduced melanoma cell line (B16-FLT3L) as
previously described22,23,38 or as a recombinant human protein.
Dendritic cells were isolated from (1) the secondary lymphoid and
GVHD target organ spleen, (2) the liver as a direct GVHD target
organ, (3) PLNs, and (4) MLNs as secondary lymphoid organs for
B
PLN
MLN
LPAM-1
Results
CFSE
Figure 1. Differential induction of homing molecules on allogeneic donor
T cells in vivo. CFSE-labeled naive allogeneic donor T cells (B10.BR) were
adoptively transferred into lethally irradiated hosts (C57BL/6), and expression of
LPAM-1, E-lig, and P-lig on donor T cells was determined in spleen, liver, PLNs, and
MLNs after 3 days. (A) Whereas the gut-homing molecule LPAM-1 is induced in
MLNs, E-lig and P-lig are induced in PLNs (PLNs vs MLNs, *P ⫽ .002, **P ⬍ .005,
***P ⫽ .001). Expression of all molecules is intermediate in spleen and liver. Error
bars represent SEM. (B) Representative example for LPAM-1 up-regulation on
dividing (CFSElo) donor T cells in MLNs.
the GVHD target organs skin and gut, respectively. The subset
composition of dendritic cells varied considerably among different
organs, except for a remarkable similarity between lymph nodes
(Figure 2A). All dendritic cells were phenotypically immature and
had high expression of MHC II (I-A/I-E) and low levels of CD80,
CD86, or CD40. Liver-derived dendritic cells had significantly
fewer MHC II–positive and CD40⫹ cells (Figure 2B,C). However,
adding LPS to our culture conditions allowed all dendritic cells to
rapidly acquire a fully mature phenotype irrespective of their
origins (Figure 2C).
Liver-derived dendritic cells have less allostimulatory capacity
In vitro stimulation with host-type dendritic cells (C57BL/6)
efficiently activated naive allogeneic donor-type T cells (B10.BR)
as evidenced by the increase of absolute cell numbers and the
acquisition of an effector memory phenotype (CD44hiCD62Llo)
with a predominance of CD8⫹ T cells (Figure 3A). This was similar
among all 4 groups. However, proliferation as determined by
3H-thymidine incorporation was lower in the liver group (Figure
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2932
A
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
KIM et al
100
75
'Lymphoid DCs' (CD11c+CD8+)
'Myeloid DCs' (CD11c+CD11b+)
'Plasmacytoid DCs' (CD11+B220+)
'Other DCs' (CD11c+CD8-CD11b-B220-)
50
25
0
B
100
Spleen
Liver
PLN
MLN
*
75
Spleen
Liver
PLN
MLN
*
50
25
0
MHC II
C
CD80
CD86
MHC II CD80
CD40
CD86
CD40
Spleen DCs
Liver DCs
homing molecules in vitro that we had previously observed to be
induced in vivo (Figure 1). Only dendritic cells from MLNs
induced LPAM-1 expression on donor CD4⫹ and CD8⫹ cells,
whereas they were significantly less capable of inducing expression
of E-lig and P-lig (Figure 4A). The induction of LPAM-1 in the
MLN group was due to significantly higher expression of both
subunits, the ␣4 and ␤7 chains. LFA-1, which has recently been
implicated in liver homing,39 was not differentially induced by
liver-derived dendritic cells. All differences were detected on
T cells with an effector memory phenotype only (data not shown).
As previously described for Peyer patch–derived dendritic cells,23
and in accordance with an active mechanism,25 MLN dendritic
cells (DCs) dominantly induced LPAM-1 and partially suppressed
the induction of E-lig and P-lig when competing with PLN DCs at
equivalent concentrations (Figure 4B). Thus, all organ-derived dendritic
cells activate allogeneic T cells but only MLN DCs up-regulate
LPAM-1 and only weakly induce E- and P-lig compared with all other
dendritic cells, thereby inducing a gut-homing phenotype.
Microarray analysis of allogeneic T cells stimulated with
different organ-derived dendritic cells reveals
differentially induced genes
PLN DCs
MLN DCs
Isotype (freshly isolated)
Isotype (overnight culture)
Costimulatory marker (freshly isolated)
Costimulatory marker (overnight culture)
Figure 2. Organ-derived dendritic cells are heterogeneous and phenotypically
immature. (A) Subset composition of organ-derived dendritic cells (CD11c⫹) defined
as lymphoid (CD8⫹), myeloid (CD11b⫹), or plasmacytoid (B220⫹) shows a large
proportion of liver-derived dendritic cells being negative for all 3 subset markers and a
high number of plasmacytoid dendritic cells in both lymph node groups (n ⫽ 14-15).
(B) Organ-derived dendritic cells are phenotypically immature, express mostly MHC II
(I-A/I-E), but only partly CD80, CD86, or CD40. Liver-derived dendritic cells have
significantly fewer MHC II–positive and CD40⫹ cells (n ⫽ 6-8, *P ⬍ .05). Error bars
represent SEM. (C) All organ-derived dendritic cells acquire a mature phenotype after
overnight culture in the presence of LPS (100 ng/mL) with further up-regulation of
MHC II and full induction of CD80, CD86, and CD40 (representative experiment).
3B) and was associated with lower levels of interleukin 2 (IL-2) in
cell culture supernatants, whereas IFN-␥ and IL-12p40 secretion
was similar (Figure 3C; Figure S3, available on the Blood website;
see the Supplemental Materials link at the top of the online article).
Expression of the activation marker CD25 was significantly lower,
whereas expression levels of CD69 did not differ from the other
organs (Figure 3D). Production of IFN-␥ and TNF-␣ when
determined by intracellular cytokine staining was similar in all
4 groups (Figure 3E). After additional stimulation with PMA and
ionomycin, expression of IFN-␥ and CD69 was comparable
(Figure S5). Regulatory T cells detected by staining for Foxp3 had
an activated phenotype (CD44hiCD62Llo, not shown) and were
found in similar numbers in all groups (Figure 3F). To determine
their suppressive capacity, we performed a suppression assay and
found that regulatory T cells from all 4 groups strongly suppressed
proliferation (Figure 3G). And although “liver” regulatory T cells
showed more suppression than the other 3 groups, this was not
statistically significant. We conclude that liver-derived dendritic
cells are less effective in the activation of allogeneic T cells.
MLN dendritic cells induce high LPAM-1 but low E- and
P-selectin ligand expression on allogeneic T cells in vitro
We next addressed whether host-derived dendritic cells from
spleen, liver, PLNs, and MLNs could differentially induce the same
To apply this culture system to discover new genes that are
differentially induced by organ-derived dendritic cells, we performed microarray analyses of these stimulated T cells using the
GeneChip Mouse Genome 430A 2.0 Array (Affymetrix). By
directly comparing 2 groups we eliminated all genes that were
equally up-regulated in both groups (eg, reflecting an activated
state). In addition, comparisons were always done between stimulated T cells from one experiment that consisted of the same
initially naive T cells with different organ-derived dendritic cells
from the same (pooled) animal. This eliminated interexperiment
variability and possible genetic heterogeneity. A first comparison
between liver and spleen was performed to identify putative
liver-homing molecules (Table 1). In the liver group, 22 genes were
up-regulated and 16 genes were down-regulated compared with the
spleen group. Two genes that have previously been implicated in
liver homing (Cxcr6 and S100a4) were found to be up-regulated. A
second comparison between MLNs with PLNs (Table 2) identified
15 and 22 different genes to be up- and down-regulated, respectively. Two genes that are induced by retinoic acid (Stra6 and
Tgm2) were up-regulated in the MLN group. Overall, there was a
major overlap in each comparison with not more than 22 of
approximately 14 000 genes being significantly up- or downregulated, indicating considerable similarities between the different
organ-derived dendritic cells and identifying potential differentially induced homing molecules.
Allogeneic T cells stimulated with MLN DCs induce the highest
GVHD mortality and morbidity
To assess the impact of different homing phenotypes on the
capacity to induce GVHD, we transplanted T cell–depleted bone
marrow and 106 stimulated T cells (B10.BR) into lethally irradiated
hosts (C57BL/6). Recipients of T cells from the MLN DC group
had the shortest overall survival (Figure 5A), with a median
survival time of 31 days as opposed to approximately
40 days for the other groups. This difference was highly statistically significant (MLN vs liver/PLN: P ⬍ .001, MLN vs spleen:
P ⫽ .001) and was also associated with higher overall clinical
GVHD scores (MLN vs spleen/PLN: P ⬍ .001, MLN vs liver:
P ⫽ .024, Figure 5B) and more gut GVHD-induced weight loss
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
Figure 3. Organ-derived dendritic cells activate naive
allogeneic T cells in vitro, but liver-derived dendritic
cells are less efficient. (A) Four days after coculture with
different organ-derived host-type dendritic cells, absolute cell
numbers of donor-type T cells are almost doubled with
preferential stimulation of CD8⫹ cells (n ⫽ 15-17). CD4⫹ and
CD8⫹ T cells express mostly an effector memory phenotype
(CD44hiCD62Llo). (B) Liver-derived dendritic cells induce
less proliferation of allogeneic T cells, as assessed by
3H-thymidine incorporation (1 representative of 4 experiments shown). (C) Determination of cytokines in cell culture
supernatant shows comparable IFN-␥ but significantly less
IL-2 secretion by T cells stimulated with liver-derived dendritic cells (n ⫽ 6, *P ⬍ .05). (D) Expression of the activation
marker CD25 is significantly lower in the liver group (n ⫽ 810, **P ⬍ .001), whereas CD69 is comparable. (E) Intracellular staining for IFN-␥ and TNF-␣ shows similar expression in
all 4 groups. (F) The induction of activated Foxp3⫹ regulatory
T cells by all organ-derived dendritic cells is comparable.
(G) The suppressive capacity of isolated CD4⫹CD25⫹ regulatory T cells is similar in a suppression assay using C57BL/6
responder and BALB/c stimulator. Panels D and F are gated
on CD44hiCD62Llo T cells. Error bars represent SEM.
DENDRITIC CELLS IMPRINT ALLOGENEIC T CELLS
A
2.5
B
100
2.0
75
1.5
25
0.5
100000
80000
CD8+
CD4+
50
1.0
60000
40000
0
0.0
2933
20000
CD4+
100
75
75
50
50
25
25
0
0
C
0
CD8+
100
50
12
40
10
4
2
0
0
E
hi
hi
lo
2000
6
10
hi
hi
D
IL-2
*
8
20
lo
CD44 CD62L
CD44 CD62L
IFN-γ
30
CD44 CD62L
1:100
1:10
CD25
**
CD69
200
1500
150
1000
100
500
50
0
0
Spleen DCs
Liver DCs
PLN DCs
MLN DCs
CD4+ CD8+
CD4+ CD8+
CD4+ CD8+
CD4+ CD8+
IFN-γ
9.45
7.93
7.73
6.51
8.73
7.46
7.83
6.26
52.5
78.9
47.2
75.5
54
79.7
52.4
80.1
4.76
6.34
5.99
7.54
4.75
7.13
4.47
5.87
54.2
77.1
53.5
80.2
59.8
82.4
56.6
82.1
TNF-α
1:1
Spleen
Liver
PLN
MLN
CD44
F
Foxp3
3
G
50000
40000
30000
2
20000
1
10000
0
+
+
+
+
+
+
+
+
+
+
+
+
Liver
PLN
MLN
+
+
Spleen
0
T cells
+
T cells + stimulators
T cells + stimulators + Tregs
(MLN vs spleen/PLN: P ⬍ .001, MLN vs liver: P ⫽ .013, Figure
5C), but not higher clinical skin GVHD scores (data not shown)
compared with the other 3 groups. This higher mortality and
morbidity correlated with increased GVHD target organ pathology
in the gut (MLN vs liver/PLN: P ⬍ .05, Figure 5D), but not in liver
or skin. At day 14, levels of IFN-␥, IL-12p70, TNF-␣, MCP-1, and
IL-10 (Figure 5E; Figure S4) and liver function tests (Figure 5F) in
the sera of mice that underwent transplantation were similar. We
did not find evidence for more skin GVHD in the PLN DC group or
more liver GVHD in the liver DC group. T cells stimulated with
MLN DCs thus induce the highest GVHD mortality and morbidity
after BMT and specifically more gut GVHD, but the absolute
difference from the other groups is rather small.
not statistically significant, we did observe more MLN DC T cells
in the gut and PLN DC T cells in the skin on days 7 and 23 (data not
shown). However, we felt that the isolation of lymphocytes from
the skin and the gut was prone to high variability. We therefore
opted for additional in situ analyses of gut-infiltrating T cells by
immunohistochemical staining and quantification of the number of
CD3⫹ cells per high-power field. On day 7, significantly higher
numbers of T cells were found in the small and large bowel of mice
that received a transplant of T cells from the MLN DC group
compared with the PLN DC group (Figure 6C). We conclude that
imprinted allogeneic T cells show differential in vivo migratory
behavior leading to preferential infiltration of the corresponding
organ of the dendritic cells that were used for stimulation.
Imprinted allogeneic T cells show differential in vivo homing
Stimulated donor T cells express homing molecules in vivo
irrespective of previous stimulation in vitro
To assess a potential liver-homing phenotype, we quantified donor
T-cell numbers in the liver at day 7 after BMT and found
reproducibly significantly higher numbers in the liver DC group
(Figure 6A). This was not due to less apoptosis as evidenced by
staining for annexin V (Figure 6B). To analyze the in vivo homing
of the PLN DC and the MLN DC group, we quantified cells in the
skin and the gut of mice that underwent transplantation. Although
One possible explanation for this moderate shift in aggressiveness
is the reprogramming of T cells that has been described previously
for in vitro and in vivo imprinted T cells23,24 and sorted unstimulated T cells.41 We therefore analyzed expression of homing
molecules on stimulated donor T cells 6 days after transplantation
in vivo (Figure 6D). Analysis of LPAM-1 and P-lig expression
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KIM et al
A
LPAM-1
*
*
25
20
B
LFA-1
PLN DCs MLN DCs
2000
1500
PLN DCs+
MLN DCs
1.4
8.8
9.5
84.2
77.1
75
37.4
21.8
23.7
28.7
56.8
59.9
62
47
46.8
22.6
39.5
40.5
15
1000
LPAM-1
10
500
5
0
CD4
*
E-lig
CD8
0
*
*
800
CD4
CD8
P-lig
*
1500
600
P-lig
1000
400
500
200
CD44
0
600
E-lig
CD4
CD8
Integrin α4
**
**
0
CD4
CD8
Integrin β1
400
300
400
200
200
100
0
CD4
CD8
Integrin β7
**
*
125
100
75
0
CD4
CD8
Spleen DCs
Liver DCs
PLN DCs
MLN DCs
50
25
0
CD4
CD8
Figure 4. Strong induction of LPAM-1, but less induction of E-lig and P-lig by MLN DCs. (A) Four days after coculture with different organ-derived dendritic cells,
expression of LPAM-1, LFA-1, E-lig, P-lig, integrin ␣4, integrin ␤1, and integrin ␤7 allogeneic T cells was analyzed. Effector memory cells (CD44hiCD62Llo) in the MLN DC group
express LPAM-1 and significantly less E-lig or P-lig (*P ⬍ .001, n ⫽ 8-15). There is also statistically significantly higher expression of integrin ␣4 and integrin ␤7 in the MLN DC
group (**P ⬍ .05, n ⫽ 5-6). Error bars represent SEM. (B) Coculture of PLN and MLN DCs at equivalent concentrations shows that MLN DCs induce LPAM-1 irrespectively of
PLN DCs, and negatively affect the induction of E-lig and P-lig (1 representative of 3 independent experiments).
showed comparable expression between the groups despite
previous stimulation with different dendritic cells. The induction of a defined homing phenotype in vitro thus appears to be
transient after adoptive transfer to an allogeneic bone marrow
transplant recipient.
Discussion
Host APCs are crucial for the initiation of GVHD, but their
possible role in imprinting homing molecules on alloreactive
donor T cells has not been addressed previously. Here, we took a
combined in vitro and in vivo approach to study the impact of
different organ-derived dendritic cells on naive allogeneic
T cells in vitro and their capacity to induce GVHD after
transplantation in vivo.
We chose to focus on the secondary lymphoid and GVHD target
organ spleen, the liver as a GVHD target organ, and the peripheral
and mesenteric lymph nodes as secondary lymphoid organs for the
GVHD target organs skin and gut. We also performed experiments
with dendritic cells from Peyer patches, which were limited by the
lower number of dendritic cells that could be obtained. However,
the main conclusions regarding MLN DCs, specifically the induction of a gut-homing phenotype and the higher mortality and
morbidity after transplantation, also apply to dendritic cells from
Peyer patches (data not shown).
A number of recent studies have shown that different organderived dendritic cells imprint a homing phenotype on naive
T cells.20-24,42 We extend these studies from transgenic autologous model
systems to an allogeneic MHC-mismatched BMT model. Whereas only
T cells stimulated with MLN DCs became positive for LPAM-1, all
T cells up-regulated their expression of E-lig and P-lig. However, the
level of E-lig and P-lig up-regulation was lower after stimulation with
dendritic cells from MLNs than after stimulation with other dendritic
cells. As previously shown for Peyer patch–derived dendritic cells,23
MLN DCs dominantly induced LPAM-1 when cocultured at equivalent
ratios with other dendritic cells and this resulted in lower expression of
E-lig and P-lig (Figure 4B).
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DENDRITIC CELLS IMPRINT ALLOGENEIC T CELLS
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Table 1. T-cell genes differentially induced by liver-derived dendritic cells
Fold change
Gene title
Gene symbol
Genebank40
Up-regulated
2,10
Regulator of G-protein signaling 16
Rgs16
U94828.1
2,09
Granzyme G
Gzmg
NM_010375.1
1,92
Secreted phosphoprotein 1
Spp1
NM_009263.1
1,89
Granzyme E* (n⫽2)
Gzme
NM_010373.1
1,77
Chemokine (C-X-C motif) receptor 6
Cxcr6
AF301018.1
1,64
S100 calcium binding protein A4
S100a4
D00208.1
1,49
Granzyme C
Gzmc
NM_010371.1
1,36
Thioredoxin interacting protein
Txnip
NM_023719.1
NM_009112.1
1,32
S100 calcium binding protein A10 (calpactin)
S100a10
1,32
Poly A binding protein, cytoplasmic 1
Pabpc1
NM_008774.1
1,29
Semaphorin 4A
Sema4a
NM_013658.1
1,29
Ribosomal protein S2* (n⫽2)
Rps2
AK004568.1
1,26
Similar to glyceraldehyde-3-phosphate dehydrogenase
LOC14433
NM_008084.1
AK002516.1
1,26
Rho, GDP dissociation inhibitor (GDI) beta
Arhgdib
1,26
Ribosomal protein, large, P1
Rplp1
NM_018853.1
1,26
Lectin, galactose binding, soluble 1
Lgals1
NM_008495.1
1,26
Ribosomal protein L5
Rpl5
BC026934.1
1,26
Ribosomal protein L26
Rpl26
NM_009080.1
1,26
Ribosomal protein L13
Rpl13
NM_016738.1
1,26
Actin, beta, cytoplasmic* (n⫽2)
Actb
M12481.1
1,26
Adenylosuccinate lyase
Adsl
K00131.1
1,26
Caspase 3, apoptosis related cysteine protease
Casp3
D86352.1
Down-regulated
⫺1,45
Calponin 3, acidic* (n⫽3)
Cnn3
BB724741
⫺1,49
Suppressor of cytokine signaling 3* (n⫽2)
Socs3
BB241535
⫺1,49
RIKEN cDNA 1110035L05 gene
1110035L05Rik
NM_026125.1
⫺1,52
Calponin 3, acidic
Cnn3
AV172168
⫺1,55
Tumor necrosis factor receptor superfamily, member 8
Tnfrsf8
NM_009401.1
⫺1,59
Interleukin 1 receptor, type II
Il1r2
NM_010555.1
⫺1,70
T-cell receptor gamma chain /// T cell receptor gamma chain
Tcrg
NM_011558.2
⫺1,74
Histocompatibility 2, Q region locus 10
H2-Q10
BC011215.1
⫺1,76
T cell receptor associated transmembrane adaptor 1
Trat1
BB622792
⫺1,78
FERM domain containing 4B
Frmd4b
BB009122
⫺1,95
Aryl-hydrocarbon receptor
Ahr
NM_013464.1
⫺2,00
Insulin-like growth factor binding protein 4* (n⫽4)
Igfbp4
BC019836.1
⫺2,45
Extracellular matrix protein 1
Ecm1
NM_007899.1
⫺3,32
Lumican
Lum
AK014312.1
⫺3,35
Interleukin 17
Il17
NM_010552.1
⫺4,61
Transglutaminase 2, C polypeptide* (n⫽4)
Tgm2
BC016492.1
Naive donor-type allogeneic T cells were cocultured for 4 days with liver-derived host-type dendritic cells, total RNA was extracted, and microarray analysis was performed
using the GeneChip Mouse Genome 430A 2.0 Array (Affymetrix). Gene expression levels were compared with T cells cultured with spleen-derived dendritic cells. Greater than
1.25-fold changes with P⬍.001 were chosen.
One potential source of bias of our results may be the use of
FLT3L to expand dendritic cells. However, the paucity in normal
organs precludes the yield of sufficient numbers of dendritic cells
for the experiments presented here and most of the previous
imprinting studies have made use of this tool. Previous reports
examining the effect of FLT3L on alloresponses have compared
FLT3L treatment with no treatment. Here, we always compared
dendritic cells that were equally exposed to FLT3L and have
therefore presumably undergone the same changes.
The liver has long been known for its unique immunologic
properties as a tolerogenic organ.43 This can be attributed in part to
its dendritic cells,44,45 and possibly to the CD8⫺CD11b⫺ dendritic
cell subset.46 We here extend the latter report, which described less
maturity and allostimulatory capacity of liver-derived dendritic
cells in a Th2-driven BALB/c into C57BL/6 model, now to a
Th1-driven B10.BR into C57BL/6 model. These results may have
been biased due to a lack of sensitivity of hepatic dendritic cells to
LPS.47 However, this was shown for a much lower concentration
than used here, and in some cases we additionally substituted LPS
with recombinant CD40L, obtaining the same results (data not
shown). Foxp3⫹CD4⫹ T cells were not increased (Figure 3F).
However, CD4⫹CD25⫹ regulatory T cells generated with liverderived dendritic cells showed a trend toward more effective
suppression in a suppression assay (Figure 3G). A number of
different molecules has been implicated in liver homing,48 such as
LFA-1,39 but we did not find significantly higher levels of LFA-1 in
the liver group. However, our microarray analysis showed induction of CXCR6 (Table 1), which has been implicated in liver
homing and GVHD.49
In contrast to previous reports using microarray analysis in
GVHD,50,51 our culture system allowed us to strictly limit our
analysis to genes directly induced in T cells after stimulation with
dendritic cells without potentially confounding parenchymal cells
or migrating T cells that have been activated in a different organ.
Most remarkable was the fact that very few genes were differentially induced. Fewer than 22 of approximately 14 000 genes
differed in each comparison (liver vs spleen, and MLN vs PLN).
Although there are significant differences between organ-derived
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Table 2. T-cell genes differentially induced by MLN-derived dendritic cells
Fold change
Gene title
Gene symbol
Genebank40
Up-regulated
4.14
Stimulated by retinoic acid gene 6
Stra6
NM_009291.1
2.37
Leucine zipper transcription factor-like 1
Lztfl1
NM_033322.1
2.00
Hypermethylated in cancer 1
Hic1
NM_010430.1
1.73
Xanthine dehydrogenase
Xdh
AV286265
1.63
Granzyme G
Gzmg
NM_010375.1
1.63
Purinergic receptor P2X, ligand-gated ion channel 4
P2rx4
AJ251462.1
1.49
Serine (or cysteine) proteinase inhibitor, clade E, member 2
Serpine2
NM_009255.1
1.49
Serine (or cysteine) proteinase inhibitor, clade A, member 3G
Serpina3g
BC002065.1
1.45
Growth factor independent 1
Gfi1
NM_010278.1
1.45
Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme
Gcnt2
BM236768
1.44
Transglutaminase 2, C polypeptide* (n⫽2)
Tgm2
BB550124
1.42
Tribbles homolog 2 (Drosophila)
Trib2
BC027159.1
1.38
Leukocyte immunoglobulin-like receptor, subfamily B, member 4
Lilrb4
U05264.1
1.35
Sprouty-related, EVH1 domain containing 2
Spred2
AV229054
1.35
GTPase, IMAP family member 3
Gimap3
NM_031247.1
Down-regulated
⫺1.26
CD44 antigen
Cd44
BC005676.1
⫺1.26
Interleukin 7 receptor
Il7r
NM_008372.2
⫺1.31
Asparagine synthetase* (n⫽2)
Asns
BC005552.1
⫺1.32
ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4
St8sia4
NM_009183.1
⫺1.39
Beta galactoside alpha 2,6 sialyltransferase 1
St6gal1
BG075800
⫺1.42
Inducible T-cell co-stimulator* (n⫽2)
Icos
NM_017480.1
⫺1.42
Membrane-spanning 4-domains, subfamily A, member 4C
Ms4a4c
NM_022429.1
⫺1.45
Selectin, lymphocyte* (n⫽2)
Sell
M36005.1
⫺1.49
Prion protein* (n⫽2)
Prnp
NM_011170.1
⫺1.50
RIKEN cDNA 1190002H23 gene* (n⫽2)
1190002H23Rik
BB408123
⫺1.56
Syndecan binding protein (syntenin) 2
Sdcbp2
BC005556.1
⫺1.61
Glutamic pyruvate transaminase (alanine aminotransferase) 2
Gpt2
BI648645
⫺1.63
Interleukin 10
Il10
NM_010548.1
⫺1.64
Palmitoyl-protein thioesterase precursor
ppt
AF087568.1
⫺1.69
Cystathionase (cystathionine gamma-lyase)
Cth
BC019483.1
⫺1.81
Interleukin 18 receptor 1
Il18r1
NM_008365.1
⫺1.95
Plexin C1* (n⫽2)
Plxnc1
BB765457
⫺2.02
Interleukin 24
Il24
AF333251.1
⫺2.11
Hemogen
Hemgn
NM_053149.1
⫺2.23
Killer cell lectin-like receptor subfamily C, member 1 /// member 2
Klrc1 /// Klrc2
AF106008.1
⫺2.25
Semaphorin 4A* (n⫽2)
Sema4a
NM_013658.1
⫺4.23
Lamin A
Lmna
NM_019390.1
Naive donor-type allogeneic T cells were cocultured for 4 days with MLN-derived host-type dendritic cells, total RNA was extracted, and microarray analysis was performed
using the GeneChip Mouse Genome 430A 2.0 Array (Affymetrix). Gene expression levels were compared with T cells cultured with PLN-derived dendritic cells. Greater than
1.25-fold changes with P ⬍ .001 were chosen.
dendritic cells, they have many similarities with respect to their effect
on T cells. We found 2 genes previously implicated in liver homing,
Cxcr649 and S100a4,52 to be induced by liver-derived dendritic cells.
Surprisingly, many granzymes were also up-regulated in the liver
compared with spleen. Although described in the setting of antitumor
activity, the full expression of an effector phenotype in vitro can lead to a
paradoxical impairment of function in vivo.53 A similar process with
respect to GVHD may apply here. The identification of 2 retinoic
acid–dependent genes induced by MLN DCs further underlines the
involvement of retinoic acid25 and identifies potential novel downstream
candidate genes in gut homing.
Several groups have shown the importance of homing and the
expression of homing molecules including CXCR3,54 CD103,55
CCR2,31 L-selectin, and ␤7 integrin56 for alloreactive donor T cells
using gene-deficient mice, blocking antibodies, or inhibitory molecules separately or in combination.29,30 We have previously shown
that specifically targeting gut homing by using either cells deficient
for the ␤7 integrin57 or sorted LPAM-1⫺ cells41 can lead to
significant improvement of GVHD mortality and morbidity. In this
study, we confirm the importance of trafficking by alloreactive
T cells to the gut: donor T cells stimulated with MLN DCs induced
more severe intestinal GVHD than T cells stimulated with other
dendritic cells and consequently higher GVHD mortality. Our data
are also in agreement with previous studies regarding the dominant
role of the gut in the pathophysiology of GVHD.5
Analysis of the in vivo homing properties confirmed the
preferential trafficking of imprinted T cells to their corresponding
organs. T cells imprinted with dendritic cells from the liver were
found in significantly higher numbers in the liver. This was most
likely due to increased homing since decreased apoptosis was not
detected. T cells imprinted with dendritic cells from the secondary
lymphoid organs PLN and MLN showed remarkable similarities in all
aspects with the exception of homing molecules that translated into a
higher degree of T-cell infiltration into the corresponding target organs
skin and gut, respectively. However, various numbers of T cells from
any group were still observed in every organ analyzed.
We observed only a delay in mortality, indicating that a
“catch-up” phenomenon is occurring. We attribute this to the
dynamic nature of imprinting as previously described in vitro.23,24
Our data suggest that this reprogramming also affects alloreactive
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
DENDRITIC CELLS IMPRINT ALLOGENEIC T CELLS
2937
Figure 5. Allogeneic T cells stimulated with host-type MLN DCs in vitro induced the highest GVHD mortality and morbidity after bone marrow transplantation in
vivo. (A) Naive allogeneic donor T cells stimulated with organ-derived host-type dendritic cells in vitro were used as donor T cells in an MHC-mismatched B10.BR into C57BL/6
murine BMT model with T cell–depleted 5 to 10 ⫻ 106 BM cells and 106 T cells (n ⫽ 28-32, 4 combined experiments). Survival was significantly shorter for the MLN DC group
(*MLN vs liver/PLN: P ⬍ .001, MLN vs spleen: P ⫽ .001) with 40 days median survival for spleen-derived dendritic cells, 39 days for liver-derived dendritic cells, 39 days for
PLN DCs, and 31 days for MLN DCs. (B) Clinical GVHD score (*MLN vs spleen/PLN: P ⬍ .001, MLN vs liver: P ⫽ .024) and (C) weight curves (*MLN vs spleen/PLN: P ⬍ .001,
MLN vs liver: P ⫽ .013) show higher GVHD morbidity for the MLN DC group. (D) Histopathologic analysis of the GVHD target organs skin, liver, and small and large bowel at
day 14. There is a trend toward higher GVHD scores in the small and large bowel of the MLN DC group (*MLN vs liver/PLN: P ⬍ .05, **MLN vs liver: P ⬍ .05). (E) Levels of
IFN-␥ and (F) liver function tests at day 14 on sera of mice that underwent transplantation are comparable among all groups. Error bars represent SEM.
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
KIM et al
A
B
*
1.0
Figure 6. Imprinted allogeneic T cells show differential in vivo homing. (A) Donor T-cell numbers in the liver
at day 7 after BMT are highest in the liver DC group
(*P ⬍ .001, n ⫽ 4-5). (B) Apoptosis as analyzed by staining for annexin V is comparable among the groups.
(C) On day 7, immunohistochemical staining and quantification of the number of CD3⫹ cells per high-power field
shows significantly higher numbers of allogeneic T cells
in the small and large bowel of mice in the MLN DC group
compared with the PLN DC group (*small bowel: P ⬍ .001,
large bowel: P ⬍ .001, n ⫽ 5). Corresponding representative photomicrographs (original magnification: ⫻ 200).
Images were visualized with an Olympus BX40 microscope (Olympus, Melville, NY) equipped with a 10⫻/0.65
aperture objective lens. Images were acquired with a JVC
digital camera GC-Qx 5HDU (JVC, Wayne, NJ). (D) The
expression of the homing molecules LPAM-1 and P-lig on
donor T cells in vivo was analyzed at day 6 after previous stimulation with different organ-derived dendritic
cells in vitro (n ⫽ 5). Both molecules are up-regulated
irrespective of previous stimulation in vitro. Error bars
represent SEM.
60
50
40
30
0.5
20
10
0
0.0
C
PLN DC
MLN DC
*
*
25
20
Spleen DCs
Liver DCs
PLN DCs
MLN DCs
15
10
5
0
D
Small Bowel
Large Bowel
P-lig+ donor T cells
LP AM-1+ donor T cells
75
40
30
Spleen DCs
Liver DCs
PLN DCs
MLN DCs
50
20
25
10
0
Spleen
Liver
0
Spleen
Liver
T cells in vivo (Figure 6D), which has previously been described to
occur within a few days in vitro.23 The use of genetically deficient
mice in previous studies of homing molecules obviously prevented
this phenomenon. Further contributing to this shift is the fact that,
although uniformly stimulated, the donor T cells were heterogeneous with a subset still having a naive phenotype (CD44loCD62Lhi).
We found no evidence for selective skin GVHD in the PLN
group. This was not completely unexpected since expression of
E-lig and P-lig was comparable in the spleen, liver, and PLN
groups. There was also no elevation of transaminases in the liver
group (Figure 5F) despite evidence for liver homing (Figure 6A),
which was most likely due to the less activated/tolerogenic state of
these T cells. Imprinting of homing molecules other than those
associated with the gut may therefore only minimally contribute to
the strikingly selective clinical involvement of GVHD target
organs.10 This is not surprising considering that the pathophysiology of GVHD includes a “cytokine storm” and a high degree of
inflammation. Highly expressed inflammatory cytokines and chemokines could be expected to “override” any effect of tissue DC
imprinting and regulate GVHD target organ damage.58 As has been
shown in numerous previous studies, T-cell polarization and
costimulation clearly also influence the degree of GVHD. As a
matter of fact, it is therefore surprising that we demonstrated any
effect of tissue DC imprinting on target organ GVHD at all.
In vivo imaging of GVHD in mice showed initial trafficking of
donor T cells to secondary lymphoid organs where the upregulation of homing molecules takes place36 followed by infiltration of target organs.37,59 We chose our experimental approach to at
least partially recapitulate this initial priming and imprinting phase
circumventing the need for trafficking to secondary lymphoid
organs. Despite an earlier report that Peyer patches are crucial for
the initiation of a graft-versus-host reaction (GVHR),60 there is
evidence that this may not apply to GVHD and that secondary
lymphoid organs may compensate for each other.61,62 Use of these
stimulated T cells in mice without secondary lymphoid organs may
further emphasize the differences in GVHD mortality.
In conclusion, by systemically analyzing the effect of different
organ-derived dendritic cells on allogeneic T cells in vitro, we found an
impact on at least 2 different levels: the degree of activation, as
exemplified by less activation by liver dendritic cells, and the imprinting
of a homing phenotype, as exemplified by the induction of LPAM-1 by
mesenteric lymph nodes. Donor T cells stimulated with dendritic cells
from the gut-associated lymphatic tissue MLNs induce significantly
higher GVHD mortality and morbidity than donor T cells stimulated
with dendritic cells from all other GVHD target organs. However, the
absence of significant target organ GVHD induced by these other
dendritic cell types indicates that previous imprinting in vitro may be
overridden in vivo. Successful targeting of homing to treat or
prevent GVHD may therefore need to be continuous and interfere with
several pathways.
Acknowledgments
This work was supported by grants HL69929, CA33049,
CA023766, CA107096, and P20-CA103694 from the National
Institutes of Health; by awards from the Leukemia and Lymphoma Society, the Ryan Gibson Foundation, the Emerald
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
DENDRITIC CELLS IMPRINT ALLOGENEIC T CELLS
Foundation, the Byrne Fund, and The Experimental Therapeutics Center of the Memorial Sloan-Kettering Cancer Center,
funded by Mr William H. Goodwin and Mrs Alice Goodwin and
the Commonwealth Foundation for Cancer Research (M.R.M.B.);
a Mildred Scheel-Stipendium of the Deutsche Krebshilfe
(T.D.K.); Deutscher Akademischer Austausch Dienst (T.H.T.);
Boehringer Ingelheim Fonds (T.H.T.); Dr Werner Jackstaedt
Stiftung (J.L.Z.); and Deutsche Forschungsgemeinschaft (J.L.Z.).
J.L.Z. is the recipient of a fellowship grant from the Lymphoma
Research Foundation. O.A. is the recipient of an Amy Strelzer
Manasevit Scholar award from the National Marrow Donor
Program (NMDP) and The Marrow Foundation.
The authors thank Ulrich H. von Andrian (The CBR Institute for
Biomedical Research) for generously providing the B16-FLT3L
cell line; Agnes Viale (MSKCC genomics core facility) for support
with the microarray analysis; Amgen for generously providing
recombinant human FLT3L; and the staff of the Laboratory of
Comparative Pathology (Cornell University) and the staff of the
2939
Research Animal Resource Center (MSKCC) for excellent assistance and excellent animal care.
Authorship
Contribution: T.D.K. designed and performed research, analyzed
data, and wrote the paper; T.H.T., J.L.Z., D.S., A.A.K., M.E.C.,
C.G.K., C.B., J.G., O.M.S., C.L., and G.F.M. performed research;
G.H. performed statistical analysis; O.A. designed research and
analyzed data; M.R.M.B. designed research, analyzed data, and
wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Marcel R. M. van den Brink, Laboratory of
Allogeneic Bone Marrow Transplantation, Memorial SloanKettering Cancer Center, Z1404, Box 111, 1275 York Ave, New
York, NY 10021; e-mail: [email protected].
References
1. Ferrara JL, Deeg HJ, Burakoff SJ. Graft-vs-host
disease. 2nd ed. New York, NY: Marcel Dekker;
1997.
2. Ferrara JL, Deeg HJ. Graft-versus-host disease.
N Engl J Med. 1991;324:667-674.
3. Shlomchik WD. Graft-versus-host disease. Nat
Rev Immunol. 2007;7:340-352.
4. Ferrara JL, Reddy P. Pathophysiology of graftversus-host disease. Semin Hematol. 2006;43:310.
5. Hill GR, Ferrara JL. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine
shields in allogeneic bone marrow transplantation. Blood. 2000;95:2754-2759.
6. Shlomchik WD. Antigen presentation in graft-vshost disease. Exp Hematol. 2003;31:1187-1197.
7. Duffner UA, Maeda Y, Cooke KR, et al. Host dendritic cells alone are sufficient to initiate acute
graft-versus-host disease. J Immunol. 2004;172:
7393-7398.
8. Shlomchik WD, Couzens MS, Tang CB, et al.
Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science.
1999;285:412-415.
9. Zhang Y, Louboutin JP, Zhu J, Rivera AJ, Emerson SG. Preterminal host dendritic cells in irradiated mice prime CD8⫹ T cell-mediated acute
graft-versus-host disease. J Clin Invest. 2002;
109:1335-1344.
10. Chakraverty R, Sykes M. The role of antigen-presenting cells in triggering graft-versus-host disease and graft-versus-leukemia. Blood. 2007;
110:9-17.
11. Zhang Y, Shlomchik WD, Joe G, et al. APCs in
the liver and spleen recruit activated allogeneic
CD8⫹ T cells to elicit hepatic graft-versus-host
disease. J Immunol. 2002;169:7111-7118.
12. Merad M, Hoffmann P, Ranheim E, et al. Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin
graft-versus-host disease. Nat Med.
2004;10:510-517.
13. Matte CC, Liu J, Cormier J, et al. Donor APCs are
required for maximal GVHD but not for GVL. Nat
Med. 2004;10:987-992.
14. Anderson BE, McNiff JM, Jain D, Blazar BR,
Shlomchik WD, Shlomchik MJ. Distinct roles for
donor- and host-derived antigen-presenting cells
and costimulatory molecules in murine chronic
graft-versus-host disease: requirements depend
on target organ. Blood. 2005;105:2227-2234.
15. Jones SC, Murphy GF, Friedman TM, Korngold
R. Importance of minor histocompatibility antigen
expression by nonhematopoietic tissues in a
CD4⫹ T cell-mediated graft-versus-host disease
model. J Clin Invest. 2003;112:1880-1886.
16. Teshima T, Ordemann R, Reddy P, et al. Acute
graft-versus-host disease does not require alloantigen expression on host epithelium. Nat
Med. 2002;8:575-581.
17. Dudda JC, Martin SF. Tissue targeting of T cells
by DCs and microenvironments. Trends Immunol.
2004;25:417-421.
18. Mora JR, von Andrian UH. T-cell homing specificity and plasticity: new concepts and future challenges. Trends Immunol. 2006;27:235-243.
19. Agace WW. Tissue-tropic effector T cells: generation and targeting opportunities. Nat Rev Immunol. 2006;6:682-692.
20. Johansson-Lindbom B, Svensson M, Wurbel MA,
Malissen B, Marquez G, Agace W. Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant. J Exp Med. 2003;198:
963-969.
21. Stagg AJ, Kamm MA, Knight SC. Intestinal dendritic cells increase T cell expression of
alpha4beta7 integrin. Eur J Immunol. 2002;32:
1445-1454.
22. Mora JR, Bono MR, Manjunath N, et al. Selective
imprinting of gut-homing T cells by Peyer’s patch
dendritic cells. Nature. 2003;424:88-93.
23. Mora JR, Cheng G, Picarella D, Briskin M,
Buchanan N, von Andrian UH. Reciprocal and
dynamic control of CD8 T cell homing by dendritic
cells from skin- and gut-associated lymphoid tissues. J Exp Med. 2005;201:303-316.
24. Dudda JC, Lembo A, Bachtanian E, et al. Dendritic cells govern induction and reprogramming
of polarized tissue-selective homing receptor patterns of T cells: important roles for soluble factors
and tissue microenvironments. Eur J Immunol.
2005;35:1056-1065.
25. Iwata M, Hirakiyama A, Eshima Y, Kagechika H,
Kato C, Song SY. Retinoic acid imprints gut-homing specificity on T cells. Immunity. 2004;21:527538.
therapeutic targets. Nat Immunol. 2005;6:11821190.
29. Sackstein R. A revision of Billingham’s tenets: the
central role of lymphocyte migration in acute
graft-versus-host disease. Biol Blood Marrow
Transplant. 2006;12:2-8.
30. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR,
Serody JS. Leukocyte migration and graft-versushost disease. Blood. 2005;105:4191-4199.
31. Terwey TH, Kim TD, Kochman AA, et al. CCR2 is
required for CD8-induced graft-versus-host disease. Blood. 2005;106:3322-3330.
32. National Center for Biotechnology Information.
Gene Expression Omnibus (GEO). http://ncbi.
nlm.nib.gov/geo/. Accessed January 2008.
33. Cooke KR, Kobzik L, Martin TR, et al. An experimental model of idiopathic pneumonia syndrome
after bone marrow transplantation: I, the roles of
minor H antigens and endotoxin. Blood. 1996;88:
3230-3239.
34. Ferrara J, Guillen FJ, Sleckman B, Burakoff SJ,
Murphy GF. Cutaneous acute graft-versus-host
disease to minor histocompatibility antigens in a
murine model: histologic analysis and correlation
to clinical disease. J Invest Dermatol. 1986;86:
371-375.
35. Hill GR, Crawford JM, Cooke KR, Brinson YS,
Pan L, Ferrara JL. Total body irradiation and
acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines.
Blood. 1997;90:3204-3213.
36. Campbell DJ, Butcher EC. Rapid acquisition of
tissue-specific homing phenotypes by CD4(⫹) T
cells activated in cutaneous or mucosal lymphoid
tissues. J Exp Med. 2002;195:135-141.
37. Beilhack A, Schulz S, Baker J, et al. In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets. Blood. 2005;106:1113-1122.
38. Shi GP, Villadangos JA, Dranoff G, et al. Cathepsin S required for normal MHC class II peptide
loading and germinal center development. Immunity. 1999;10:197-206.
26. Sigmundsdottir H, Pan J, Debes GF, et al. DCs
metabolize sunlight-induced vitamin D3 to ‘program’ T cell attraction to the epidermal chemokine
CCL27. Nat Immunol. 2007;8:285-293.
39. Sato T, Habtezion A, Beilhack A, Schulz S,
Butcher E, Thorlacius H. Short-term homing assay reveals a critical role for lymphocyte functionassociated antigen-1 in the hepatic recruitment of
lymphocytes in graft-versus-host disease.
J Hepatol. 2006;44:1132-1140.
27. Mora JR, Iwata M, Eksteen B, et al. Generation of
gut-homing IgA-secreting B cells by intestinal
dendritic cells. Science. 2006;314:1157-1160.
40. National Center for Biotechnology Information.
GenBank. http://ncbi.nlm.nih.gov/GenBank/. Accessed January 2008.
28. Luster AD, Alon R, von Andrian UH. Immune cell
migration in inflammation: present and future
41. Petrovic A, Alpdogan O, Willis LM, et al. LPAM
(alpha 4 beta 7 integrin) is an important homing
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2940
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
KIM et al
integrin on alloreactive T cells in the development
of intestinal graft-versus-host disease. Blood.
2004;103:1542-1547.
42. Dudda JC, Simon JC, Martin S. Dendritic cell immunization route determines CD8⫹ T cell trafficking to inflamed skin: role for tissue microenvironment and dendritic cells in establishment of T cellhoming subsets. J Immunol. 2004;172:857-863.
43. Crispe IN. Hepatic T cells and liver tolerance. Nat
Rev Immunol. 2003;3:51-62.
44. Lau AH, Thomson AW. Dendritic cells and immune regulation in the liver. Gut. 2003;52:307314.
CXCR6 in recruitment of activated CD8⫹ lymphocytes to inflamed liver. J Immunol. 2005;174:
277-283.
50. Ichiba T, Teshima T, Kuick R, et al. Early changes
in gene expression profiles of hepatic GVHD uncovered by oligonucleotide microarrays. Blood.
2003;102:763-771.
51. Sugerman PB, Faber SB, Willis LM, et al. Kinetics
of gene expression in murine cutaneous graftversus-host disease. Am J Pathol. 2004;164:
2189-2202.
45. Thomson AW, Lu L. Are dendritic cells the key to
liver transplant tolerance? Immunol Today. 1999;
20:27-32.
52. Taylor S, Herrington S, Prime W, Rudland PS,
Barraclough R. S100A4 (p9Ka) protein in colon
carcinoma and liver metastases: association with
carcinoma cells and T-lymphocytes. Br J Cancer.
2002;86:409-416.
46. Pillarisetty VG, Shah AB, Miller G, Bleier JI, DeMatteo RP. Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition. J Immunol.
2004;172:1009-1017.
53. Gattinoni L, Klebanoff CA, Palmer DC, et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of
adoptively transferred CD8⫹ T cells. J Clin Invest. 2005;115:1616-1626.
47. De Creus A, Abe M, Lau AH, Hackstein H, Raimondi G, Thomson AW. Low TLR4 expression by
liver dendritic cells correlates with reduced capacity to activate allogeneic T cells in response to
endotoxin. J Immunol. 2005;174:2037-2045.
54. Duffner U, Lu B, Hildebrandt GC, et al. Role of
CXCR3-induced donor T-cell migration in acute
GVHD. Exp Hematol. 2003;31:897-902.
48. Sato T, Thorlacius H, Butcher E. Lymphocyte
Homing to the Liver. Curr Med Chem - Anti-Inflammatory Anti-Allergy Agents. 2004;3:331-339.
55. El-Asady R, Yuan R, Liu K, et al. TGF-{beta}-dependent CD103 expression by CD8(⫹) T cells
promotes selective destruction of the host intestinal epithelium during graft-versus-host disease. J
Exp Med. 2005;201:1647-1657.
49. Sato T, Thorlacius H, Johnston B, et al. Role for
56. Dutt S, Ermann J, Tseng D, et al. L-selectin and
beta7 integrin on donor CD4 T cells are required
for the early migration to host mesenteric lymph
nodes and acute colitis of graft-versus-host disease. Blood. 2005;106:4009-4015.
57. Waldman E, Lu SX, Hubbard VM, et al. Absence
of beta7 integrin results in less graft-versus-host
disease because of decreased homing of alloreactive T cells to intestine. Blood. 2006;107:
1703-1711.
58. Chakraverty R, Cote D, Buchli J, et al. An inflammatory checkpoint regulates recruitment of graftversus-host reactive T cells to peripheral tissues.
J Exp Med. 2006;203:2021-2031.
59. Panoskaltsis-Mortari A, Price A, Hermanson JR,
et al. In vivo imaging of graft-versus-host-disease
in mice. Blood. 2004;103:3590-3598.
60. Murai M, Yoneyama H, Ezaki T, et al. Peyer’s
patch is the essential site in initiating murine
acute and lethal graft-versus-host reaction. Nat
Immunol. 2003;4:154-160.
61. Welniak LA, Kuprash DV, Tumanov AV, et al.
Peyer patches are not required for acute graftversus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation. Blood. 2006;107:410-412.
62. Beilhack A, Schulz S, Baker J, et al. Prevention of
acute graft-versus-host disease by blocking T-cell
entry to secondary lymphoid organs. Blood. Prepublished on November 7, 2007, as DOI 10.1182/
blood-2007-09-112789.
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2008 111: 2929-2940
doi:10.1182/blood-2007-06-096602 originally published
online January 4, 2008
Organ-derived dendritic cells have differential effects on alloreactive T
cells
Theo D. Kim, Theis H. Terwey, Johannes L. Zakrzewski, David Suh, Adam A. Kochman, Megan E.
Chen, Chris G. King, Chiara Borsotti, Jeremy Grubin, Odette M. Smith, Glenn Heller, Chen Liu,
George F. Murphy, Onder Alpdogan and Marcel R. M. van den Brink
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